key: cord-022779-himray6q authors: nan title: Abstracts of oral presentations date: 2005-06-10 journal: Biopolymers DOI: 10.1002/bip.20321 sha: doc_id: 22779 cord_uid: himray6q nan S. Tchertchian, F. Oplinger, M. Paolini, S. Manganiello, S. Raimondi, B. Depresle, N. Dafflon, H. Gaertner, and P. Botti GeneProt Inc., Geneva Branch, 2, Pré de-la-Fontaine, 1217 Meyrin, Switzerland The last decade has provided extensive demonstration of the key role played by Native Chemical Ligation (NCL) for the preparation of small and medium size proteins [1] . Yet the requirement for cysteine at the site of ligation in standard NCL has limited its flexibility. Recently, different types of auxiliary groups [2, 3] have been developed to extend the application of NCL to other ligation sites. However, the generally slower ligation rates especially with large fragments and the additional step required to cleave the auxiliary post-ligation have reduced their utility. Here we present a novel strategy to synthesize proteins through a chemical ligation using unprotected peptide segments. Our scheme does not make use of auxiliary groups [2, 3] , instead originally exploits the features of some side chain removable functionalities. Ligation rates are high, comparable to NCL and the residues available for ligation are more frequent than cysteine. Furthermore the whole process is "one pot" and at the end a native polypeptide is obtained directly in the ligation mixture. The total chemical syntheses of C5a (1-74) using both NCL and our method will be presented and compared. [ During the biosynthesis of glycopeptide antibiotics of the vancomycin family, several oxidative phenol coupling reactions take place. The enzymes catalyzing these reactions are of interest from structural and mechanistic viewpoints. In this work [1, 2] , it is shown that the oxygenase OxyB from the vancomycin producer only catalyzes a phenol coupling reaction when the putative peptide substrate is linked as a thioester to a peptide carrier domain (PCD) derived from the nonribosomal peptide synthetase. An efficient access is described to representative free linear peptide substrates, which makes use of Alloc-solid phase peptide chemistry, but largely avoids the use of amino acid side chain protecting groups. In this way, the target linear peptides can be released from the resin under very mild conditions, and then be activated as thioesters, prior to loading onto the PCD. [ We have recently discovered a new nonenzymatically-formed product from N-(3-oxododecanoyl)-L-homoserine lactone. Interestingly, both the N-acylhomoserine and its novel tetramic acid degradation product 1 are potent antibacterial agents. Bactericidal activity was observed against all tested Gram-positive bacterial strains, while no toxicity was seen against Gram-negative bacteria. We propose that P. aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. Additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization. Using short portions (7-11 amino acid) rich in positively charged residues from either human lactoferricin or the MARCKS protein as templates, a panel of 70 peptides each possessing a specific chemical structure was synthesized. These included amino acid omissions, substitutions, and insertions in the aim to modify the peptides overall charge, hydrophobic core, and/or amphipathicity. The peptides antimicrobial activities against a large panel of bacteria were assessed using both conventional tests (MIC, MBC) and non-conventional assays (MIC quantified by an automated turbidimetry-based system and MBC measured on resting cells suspended in low-ionic strength medium-"survival assay"). Furthermore, the membrane permeabilizing activity of the peptides on strains of several Gram negative bacterial species was quantitated by measuring their ability both to decrease the MIC of novobiocine and to promote the uptake of the hydrophobic fluorescent probe NPN. While the MIC determined by turbidimetry or by the conventional method did not significantly differ, bactericidal activity of the peptides measured by the survival assay was 1 to 2 orders of magnitude higher than that measured by the conventional MBC test. On the other hand, the two assays used to measure the permeabilizing activity of the peptides rendered similar results. Interestingly, the most potent permeabilizers did not correspond with the peptides exhibiting the highest bactericidal activity thus indicating that these two activities have different structural bases. Protein farnesyl transferase (PFTase) catalyzes the attachment of farnesyl diphosphate (FPP) to proteins that contain a CAAX-box sequence at their C-termini [1] . Several analogues of FPP that incorporate azide functional groups have been synthesized and shown to be incorporated into peptides using PFTase as a catalyst. In particular, it has been shown that the prenyl azide moiety from 1 or related analogues can be transferred to the peptide substrate, N-dansyl-GCVIA to yield the corresponding thioether-linked products. The resulting azide-containing peptides have been derivatized with a triphenylphosphine-based reagent to generate O -alkyl imidate-linked products rather than the amide-linked material expected via a Staudinger reaction [2] . Since CAAX-box sequences can be appended to the C-termini of many different proteins, these analogues provide a simple and general method for incorporating orthogonal azides into proteins at unique sites. Subsequent functionalization of such azide groups via Staudinger or "Click" chemistry should provide a convenient method for linking proteins with a diverse array of probes, biomolecules, surfaces and other materials under mild conditions. Chemoselective glycosylation, acylation, and alkylation of completely unprotected peptides can be accomplished by incorporating N-alkylaminooxy amino acids into the peptide sequence. The N-alkylaminooxy side chains react selectively with reducing sugars, activated alkyl halides, and various acylating agents in mildly-acidic aqueous buffers (pH 4) to furnish neoglyco-and neolipopeptides. A key feature of the approach is that a single parent peptide can be quickly reacted with a variety of agents to provide a large number of "post-translationally"modified peptides. The ability to easily synthesize arrays of modified peptides allows comprehensive studies of the effects that glycosylation and lipidation have on peptide structure and function. Here we present an overview of the methodology and initial results on its application to studying problems of biological interest. This presentation describes cyclic peptides that fold into well-defined ␤-sheet structures in aqueous solution and can dimerize through ␤-sheet interactions. The cyclic peptides contain the unnatural amino acid Hao, which mimics the hydrogen-bonding pattern of one edge of a peptide ␤-strand, and ␦-linked ornithine, which mimics a ␤-turn and provides enhanced water solubility or a linkage point for creating multivalent structures. Institute for Molecular Bioscience, The University of Queensland, Queensland 4072, Australia The Human Genome Project and other major sequencing projects have rapidly provided a vast array of new protein/peptide sequences. In contrast, many other new proteins/peptides are also being uncovered from plant and animal sources whose genomes are yet to be tapped. In the post-genomic era, the physical form of many of these gene-encoded sequences will be vital for biomedical research and drug development. Moreover, the advantages of peptide and protein chemical synthesis over recombinant-DNA methods are increasingly being used to provide rapid structure-activity information of complex bioactive peptides, small proteins and functional receptor domains. In a program designed to exploit the potential of Australian Conus species we have isolated, characterised and chemically synthesised a wide range of novel conotoxins. These cysteine rich microproteins have well-defined tertiary structures with considerable rigidity and stability and contain many elements of protein secondary structure. Of particular interest are the two disulfide bond containing conotoxins (examples below) which target transporters, ion channels and receptors at nanomolar potencies. In this presentation I will describe some of our research on controlling the shape and potency/selectivity of these microproteins through intramolecular native ligation chemical approaches. It appears that there is considerable scope to control the properties of these native sequences which in some cases may prove useful in the development of these molecules as therapeutic candidates. Despite identical amino acid composition, differences in the properties of class A amphipathic helical peptides due to differences on the hydrophobic face results in substantial differences in anti-inflammatory properties. One of these peptides is an apolipoprotein A-I mimetic, D-4F. When given orally to mice and monkeys, D-4F caused the formation of pre-␤ HDL, improved HDL-mediated cholesterol efflux, reduced lipoprotein lipid hydroperoxides, increased paraoxonase activity and converted HDL from pro-inflammatory to anti-inflammatory. In apoE null mice D-4F increased reverse cholesterol transport from macrophages. Oral D-4F reduced atherosclerosis in apoE null and LDL receptor null mice. In vitro D-4F caused the formation of pre-␤ HDL, reduced lipoprotein lipid hydroperoxides and converted HDL from pro-inflammatory to anti-inflammatory. Physical properties and the ability of various class A amphipathic helical peptides to activate enzyme LCAT in vitro did not predict biologic activity in vivo. In contrast, the use of cultured human artery wall cells in evaluating these peptides was more predictive of their efficacy in vivo. Thus, anti-inflammatory properties of different class A amphipathic helical peptides depends on subtle differences in the configuration of the hydrophobic face of the peptides. Physical-chemical properties provide an explanation for the mechanism of action of the active peptides. Peptides to ameliorate atherosclerosis and other inflammatory diseases can be designed using this strategy. inflammatory diseases. This chemokine belongs to the family of CXC chemokines, its response is mediated through binding to seven transmembrane helical G-protein coupled receptors CXCR1 and CXCR2. In order to investigate the relevance of selected protein segments for biological activity we synthesized chemically modified and biologically active analogues of the 77-mer of hIL-8 by expressed protein ligation (EPL). For ligation naturally occurring cysteine at position 55 was chosen. C-terminal peptides carrying an N-terminal cysteine were synthesized by solid phase peptide synthesis (SPPS) applying the Fmocstrategy and used to introduce modifications. Ligation of the recombinantly produced thioester with synthetic peptides yielded in full length hIL-8 that finally was correctly folded and stabilised by two disulfide bridges as in the native protein. In addition to fluorescent and photoactivatable analogues, we produced variants that contain a ␤-peptide helix instead of the naturally occurring ␣-helix. Thus, for the first time, we received a protein containing a whole ␤-peptide segment and still showing high biological activity. Depending on the linker between ␤-sheet and ␤-peptide helix of interleukin 8 we could discriminate between active and inactive proteins suggesting that the overall orientation of the C-terminal segment is highly relevant for the folding of the protein and subsequently for the signalling of interleukin 8. A peptide based on residues 109 -122 of the Syrian hamster prion protein (H1) forms ␤-sheet aggregates in solution, which grow to form large fibers. Isotope-edited infrared spectroscopy has shown that the initial antiparallel ␤-sheet formed by this peptide is disordered. A slow rearrangement occurs to form a structure in which the hydrophobic core of the strands (residues 112 -122) pack together, resulting in the alignment of residue 117 across the sheet. The kinetics of the realignment have been monitored for H1 and for peptides with mutations at residue 117 (A117I, A117L and A117B where B is aminobutyric acid). H1 and A117I align with non-exponential kinetics. At low concentrations H1 aligns via the repeated detachment and annealing of strands, whereas at higher concentrations a reptation mechanism is observed. A117B aligns instantaneously within the dead-time of our experiments. A117L does not align at residue 117 but some undefined reordering can still be observed as a shift of the 13 C band. These data are the first experimental probes of the types of intersheet rearrangements which are required for the nucleation of fibrous peptide aggregates, and the evidence for strand reptation within the ␤-sheet confirms observations in molecular dynamics simulations. [1, 2] . We have since established the microfluidic peptide chip as a miniaturizing platform. The challenging issues in making peptide chips a practical tool for understanding biology, drug discovery, and diagnostics are quality of synthesis, specificity in reported activities, and ability for quantitative measurements. We will present the results of the work which involves our intensive effort in: • Development of the method for monitoring and analyzing quality of peptide chip synthesis • Improvement in peptide chip synthesis • Development of the methods for quantitative analysis of (a) the specific binding of antibodies/proteins to peptides on chip and (b) kinase enzymatic activities against substrate peptides on chip. Our presentation should demonstrate novel applications of peptide chips that can be implemented as routine laboratorial processes. [1] Gao, X., Pellois, J. P., Kim, K., Na, Y. , Gulari, E., and Zhou, X. To prototype this approach we developed a protein array consisting of the Ras-binding domain of cRaf-1 (RBD) that was C-terminally modified with a 24mer oligonucleotide and immobilized via hybridization with the complementary oligonucleotide on a wafer [1] . The RBD-DNA conjugate was generated by native chemical ligation using a recombinantly produced RBD-thioester and an oligonucleotide carrying a 5'-cystein residue [2] . Incubation of the immobilized RBD with Ras(GTP), the activated RBD-binding form, retains sufficient amounts of Ras on the protein array to allow detection by mass spectrometry with high sensitivity. Controls carried out with inactive Ras(GDP) did not produce any signal, demonstrating a sufficient selectivity for biotechnological applications. Protein microarrays in which proteins are immobilized to a solid surface are ideal reagents for high-throughput experiments that require very small amounts of analyte. Such protein microarrays ('protein chips') can be used very efficiently to analyze all kind of protein interactions en masse. The present work describes a general method for the selective attachment of proteins to solid surfaces through its C-termini that can be used for the creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal ␣-thioester and a modified surface containing N-terminal Cys residues. ␣-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing modified inteins. We also present an efficient solid-phase approach for the rapid synthesis of Cys-containing linkers that can be used for the modification of Au-and Si-based surfaces. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain. A series of glycopeptides based on the Leu-enkephalin analogue Yt-GFS*-CONH 2 led to greatly enhanced stability in vivo and effective penetration of the BBB. Transport through the BBB hinges on the biousian nature of the glycopeptides-the glycopeptides have two conflicting conformational manifolds, a H 2 O soluble state, and an amphipathic state at H 2 O-membrane phase boundaries. Multiple lines of evidence suggest that the BBB transport mechanism is absorptive endocytosis. Mixed /␦-agonists showed antinociceptive potencies greater than morphine, and lacked many of the side effects generally associated with classical -selective opiate analgesics. The biousian design was extended to larger glycopeptides (16 residues) related to ␤-endorphin, which also penetrated the BBB and produced antinociception in mice. Plasmon waveguide resonance (PWR) studies showed that the amphipathic helices bound to membrane bilayers with M to low nM K D 's. The presence of diverse endogenous neuropeptide transmitters and neuromodulators in the human brain is potentially applicable to the treatment of a wide range of behavioral disorders. Clemencia Pinilla, 1 Mireia Sospedra, 2 Yindong Zhao, 3 We have recently demonstrated the feasibility of utilizing the ligase activity of inteins for the in vivo backbone cyclization of peptidic chains. This procedure -called SICLOPPS for Split Intein Circular Ligation Of Peptides and ProteinS-provides a biosynthetic pathway for peptides that are metabolically stable, and can be produced with spatial and temporal control [1] . To screen for bacteriotoxic peptides, a SIC-LOPPS library was introduced into an Escherichia coli population, such that each bacterium encodes a different peptide sequence. SICLOPPS library over-expression afforded six distinct bacteriostatic peptides that reduce cell growth. One of these peptides (LN05) also caused cell aggregation. An E. coli genomic library was introduced into cells encoding LN05. Co-expression of the genomic library and LN05 peptide rescues growth only in cells expressing genomic fragments able to counteract peptide toxicity. Genomic library and LN05 co-expression resulted in enrichment of a single genomic construct, a fragment of the NarZ gene. NarZ is part of a nitrate reductase complex and has a role in tuberculosis persistence [2] . LN05 production in Mycobacterium smegmatis resulted in a slow-growth phenotype. [1] Abel-Santos, E., Scott, C. P., and Benkovic, S. J., Methods in The special feature of proteins involved in Alzheimer's or prion diseases is their ability to adopt at least two different (meta) stable conformations. Thus, amyloid-forming proteins that mainly contain ␣-helical structures in their native conformation must undergo an ␣-helix3␤strand conversion before or during fibril formation. The conformational transition that shifts the equilibrium from the functional to the pathological isoform can happen sporadically. It can also be triggered by mutations in the primary structure, changes of the environmental conditions such as pH, ionic strength, metal ions, protein concentration, oxidative stress, free radicals, action of physiological, or pathological chaperones. Alternatively, the introduction of a small quantity of protein polymer may act as a structural template and initiate the disease. Therefore, the development of model systems which allow the investigation of the complex folding mechanisms that lead to ␤-sheet aggregation appears to be one of the main challenges in the detailed understanding of the pathways from incubation to mortality. In order to create an ␣-␤ switch system we designed ideal ␣-helical parallel coiled coil peptides, introduced trigger functions by mutations in the primary structure, and studied the consequences that these mutations have on the secondary structure properties of the resulting peptides under certain environmental conditions. Based on these results we continued to change the primary structure of the coiled coil system subsequently by mutations in the heptad repeat untill we ended up with soluble ␤-sheet peptides. The most important feature of these new ␤-sheet peptides is that they still follow the characteristic hydrophobic heptad repeat of an ␣-helical coiled coil and that all of the positions which are part of the dimerization domains of the coiled coil remained untouched. Thus, these new peptides bear all of the requirements which are necessary for formation of cooperatively interacting helical structures and, furthermore, contain domains for cooperative sheet aggregation as well. The folded structure will now strongly depend on the environment. This peptidic model system allows a systematic study of the subtle influences that environmental conditions may have on protein folding stepwise, which means changing these conditions one after one or in all of the possible combinations that nature applies in vivo. Plasmon Waveguide Resonance (PWR) spectroscopy is a powerful new biophysical method which allows us to examine structural changes, kinetics and thermodynamics of anisotropic biological systems and processes such as proteolipid membranes. This method has probed the mechanisms of G-protein coupled receptor (GPCR) signal transduction, and has obtained new insights into specific signaling pathways of agonists and antagonists with GPCRs. We now have extended these studies to examine the effects of lipid microdomains (rafts) on the binding, signaling and transduction pathways. Using a 1:1 mixture of palmitoyloleoylphosphatidyl-choline (POPC) and sphingomyelin (SM), we have directly observed the formation of two lipid bilayer microdomains, and the preferred segregation of the delta opioid receptor (hDOR) into SM lipid rafts when the agonist ligand was bound, but not for the unoccupied receptor which preferentially incorporated into the POPC-rich domain. Furthermore, we can demonstrate directly that, G-proteins bind much more strongly to the hDOR receptor in the lipid raft (SM-rich) environment than in the fluid non-raft (POPC-rich) domain of the lipid bilayer. The implications of these findings for novel design of drugs, and drug screening will be emphasized. † Supported by grants from the U.S.P.H.S., National Institute of Drug Abuse and National Science Foundation. Their measurement in high resolution NMR requires partial molecular alignment. For proteins in aqueous solution a number of standard methods exist to achieve such small alignment. We found that swelling of cross-linked polymers inside the NMR tube results in anisotropic gels. Peptides in such a gel phase exhibit well resolved spectra of the partially aligned molecules. This allows to scale the orientation depending on the cross-linking of the polymer, the thickness of the unswollen stick or the temperature. RDCs can now easily be measured in natural abundance in peptides 3] all common NMR solvents can be used. With the chiral gel gelatin it is possible to discriminate D-and L-Alanine to determine enantiomeric purity. The procedure is demonstrated to refine the solution structures of peptides such as cyclosporin A, somatostatin analogues and others Galia Blum, 1 Georges von Degenfeld, 2 Kinneret Keren, 3 Misregulation of cysteine protease activity is associated with numerous pathologies ranging from cancer to autoimmune disease. Protease activity is controlled by a delicate balance of many factors such as levels of natural inhibitors and posttranslational modifications. Thus developing a detection method for monitoring protease activity rather than abundance is desirable. Here we describe the design and synthesis of a novel class of chemical tools, activity based probes (ABPs), that detect protease activity. These probes are composed of a fluorescent tag and its cognate quenching group, a peptide recognition scaffold, and a reactive "warhead". These fluorescently quenched "smart probes" covalently modify protease active sites in a fashion that is dependent on activity of the protease. This results in loss of the quenching group, producing a fluorescent signal. We report the production of selective, cell permeable activity based probes for the study of papain family cysteine proteases in cells and whole animals. These probes are used to monitor real time protease activity in living cells using fluorescence microscopy techniques as well as standard biochemical methods.