key: cord-0892122-x5g7g2xu
authors: Munusamy, Sathishkumar; Conde, Renaud; Bertrand, Brandt; Munoz-Garay, Carlos
title: Biophysical approaches for exploring lipopeptide-lipid interactions
date: 2020-01-21
journal: Biochimie
DOI: 10.1016/j.biochi.2020.01.009
sha: a806a37a43e32e4d948f2a6f7567bbbb7f94b8d3
doc_id: 892122
cord_uid: x5g7g2xu

In recent years, lipopeptides (LPs) have attracted a lot of attention in the pharmaceutical industry due to their broad-spectrum of antimicrobial activity against a variety of pathogens and their unique mode of action. This class of compounds has enormous potential for application as an alternative to conventional antibiotics and for pest control. Understanding how LPs work from a structural and biophysical standpoint through investigating their interaction with cell membranes is crucial for the rational design of these biomolecules. Various analytical techniques have been developed for studying intramolecular interactions with high resolution. However, these tools have been barely exploited in lipopeptide-lipid interactions studies. These biophysical approaches would give precise insight on these interactions. Here, we reviewed these state-of-the-art analytical techniques. Knowledge at this level is indispensable for understanding LPs activity and particularly their potential specificity, which is relevant information for safe application. Additionally, the principle of each analytical technique is presented and the information acquired is discussed. The key challenges, such as the selection of the membrane model are also been briefly reviewed.

The continuous emergence and spread of multi-resistant human pathogenic bacteria require new antibacterial agents. Biosurfactants (Bs) are promising candidates to address this issue. In the past decades, a wide variety of Bs has been identified, including glycolipids, polysaccharides, proteins and lipopeptides (LPs) [1] . Amongst them, the extensive research on LPs has been fueled by their antimicrobial, antitumor, immunosuppressant and surfactant activities [2] . LPs are amphiphilic in nature, with the general structure of one or more lipid chains attached to a short linear or cyclic oligopeptide. LPs cause cell death by forming pores in the cell membranes, leading to an imbalance in transmembrane ion fluxes [3] . Hence, there is a growing interest among the scientific community to discover new LPs and implement both old and new ones for diverse environmental and pharmaceutical applications [4] . This review focuses on the analytical techniques used to characterize the interaction of LPs with biological membranes. In order to set the bases of LPs science, we present a brief overview of these topics. This review aims at introducing the main analytical techniques used to analyze the interaction and the distorting effect of LPs with artificial membranes. Some examples of the methods used to identify the interaction between lipids and LPs are also discussed.

Antimicrobial LPs have been identified and isolated from a wide range of life forms, including plants, animals and microorganisms. However, due to their abundance, ease of production and purification, bacterial antimicrobial LPs are the most studied LPs. Antimicrobial LPs are either cationic or anionic, with antimicrobial properties against gram-negative or gram-positive bacteria, respectively (Table 1) .

Recent research on the biochemical and biophysical studies of membrane-active peptides are aimed at the elucidation of the mechanism of their biological activity. These studies have led to the design of synthetic cationic peptides. Synthetically developed cationic LPs are significantly smaller than their natural counterparts [22e24] . Considerable studies on LP structure-activity relationships have resulted in the identification of salient features of effective cationic LPs. Synthetic LPs include semisynthetic LPs [25] , LPs originating from lactoferrin [26] , acylated D LPs, L-amino acid (AA) containing antimicrobial peptides (AMPs) [27, 28] , LPs based on dermaseptin [29] , extremely short LPs and peptidomimetics [30] . The detailed discussion of these LPs has been made by Roman et al. [31] .

Bacterial and fungal membranes are the main targets of LPs, though these may also interfere with other important cellular processes such as DNA replication, transcription and translation [32, 33] . In the case of the bacterial membranes, LP insertion can lead to multiple defects in the cell, such as dissipation of the transmembrane potential and hindrance in ATP formation; altogether resulting in rapid death [34, 35] . LPs have unique structural composition and function, differing from most of the other antimicrobial agents. Their antimicrobial activity is mainly due to membrane potential depolarization, occurring by direct binding to the bacterial membrane [27, 36, 37] . Also, their lability promotes a rapid elimination of the antibiotic from the environment, thereby preventing resistance development. Cationic LPs act through a 'selfpromoted uptake' mechanism, which introduces defects into the packing of lipid moieties of the outer membrane layer. Cationic LPs can neutralize endotoxins from gram-negative bacteria [38, 39] . Furthermore, acylation of LPs results in increased affinity for the target membrane.

Divalent cations, such as Ca 2þ , play a key role in the antimicrobial activity of a number of LPs. By coordination, Ca 2þ locks the bioactive molecule into an active conformation. The Ca 2þ ions also induce micelle formation, which interact with bacterial membranes [35] . The lipid tail plays an important role in its insertion of LPs, regardless of the net charge of the moiety [40] .

The common structure of naturally expressed LPs is a cyclic head group attached to a single lipid chain ( Table 2 ). The absence of free C-and N-termini in the cyclized form of the peptide unit enhances it's in vivo stability, due to reduced proteolysis [41] .

Another class of LPs are constituted by a linear peptide head group attached to one, two or three hexadecyl (palmitoyl) lipid chains. These LPs are usually produced synthetically, based on bioderived sequences [44, 45] . A lipopeptide can only be commercially viable when they have suitable production and economic recovery procedures, as well as down stream isolation protocols. In recent years, efficient and high throughput processing methods have been developed for the isolation and purification of commercial LPs [46] . The methods frequently used for LPs purification are solvent extraction, ammonium sulfate precipitation, ultrafiltration, and dialysis. Detailed discussion of these methods can be found elsewhere [47e54]. Tsushima [5] Polymyxin B related Gentamycin [6, 7] Colistin [17, 18] Aspartic

[8e10] Octapeptin [19] Laspartomycin [11, 12] Lipopeptaibols group Surfactin

[13e15] Trichogin [20, 21] Cystallomycin [16] Daptomycin [11] 1.2. The use of model lipid membranes in LPs-lipid interaction studies

Cell membranes are complex, dynamic and diverse systems, since they contain wide variety of lipid types and are crowded with different proteins, endowing them with the plasticity needed to full fill their key cellular roles [55e57] . The intrinsic heterogeneous complexity of cell membranes is still poorly understood, and thus, systematic studies of their processes and structure are impractical. As a consequence, a large number of different simplified model systems have been developed over various decades that reduce their natural complexity while mimicking their essential structural and chemical aspects, at the same time providing an easily accessible experimental platform [58, 59] . In order to conserve these structural and chemical aspects as much as possible, properties such as the proportions of the major lipids, proportions of saturated and unsaturated lipids, length of the lipid hydrocarbon chain, polar head net charge, the presence of very particular lipids of the organism and representative sterols of the taxonomic kingdom, must be taken into account [60] . The most commonly used models as we shall see further on includes Langmuir monolayers, vesicles, black lipid membranes (BLM), and solid supported bilayer lipid membranes (SLBs) [58] .

In the last couple of decades, there has been a dramatic evolution in our understanding of cell membranes and the principles that govern their behavior, as experimental techniques are becoming more sophisticated. Experimental techniques and theoretical advances include improved methods for single-molecule tracking, fluorescence correlation spectroscopy, super-resolved imaging, scattering, solid-state NMR, mass spectrometry and molecular dynamics, as well as methods to prepare asymmetric model membranes and real cell membrane extracts [57, 61] .

In any case, these simplified lipid models used for investigating the factors that affect the activity of these biomolecules and the description of their mechanism of action should be "validated". In this sense, validation refers to clearly comparable tendencies (in activity and specificity of membrane active biomolecules) between data obtained using lipid membrane models and experimental data obtained with the target cells of interest. For instance, in our investigation group, we generally use liposome models and quantify the activity and specificity of membrane active peptides by liposome leakage experiments. Once the models are "validated" they can then be used as a platform for thoroughly carrying out biophysical characterization of membrane active bio molecules like LPs, using some of approaches reviewed here. Important to note is that although the tendencies between model membranes and assay with cells may be similar, the effective concentration range may vary [62] . Let alone, the study of cell membranes is complex, and even more the study of the effects that biological agents have on them. Hence, constant improvements of these models are required. The aim of using lipid membrane models is to better understand the mechanism of action of membrane active biomolecules before moving to the complex biological systems. The final conclusions of the mechanisms by which LPs achieve specificity towards a particular membrane will always ultimately require corroborations in the target organism.

The primary structure of a large number of LPs has been elucidated. As LPs have no primary structure homology, they are characterized by their cationic/anionic and hydrophobic properties. The principal secondary structure of most LPs is a-helical, although some b-sheet lipopeptides also have antimicrobial activity. In some cases, a-LPs, which have been modified to possess b-sheet retain part of their antimicrobial activity. Even though cationic LPs have structural diversity, their amphipathic nature allows them to interact with the membrane interface.

Among the structural features of LPs, self-assembly is touted to be an important characteristic of their antimicrobial activity. Above the Critical Micelle Concentration (CMC), LPs self-assemble into spherical micelles or other structures (nano fibrils or nano tapes). Most LPs exhibit self-assembly at millimolar concentration ranges. At this concentration, the membrane rupture effect of LPs is evident. For example, it has been found that the antimicrobial property of daptomycin is a cooperative phenomenon above a threshold concentration [63] . Moreover, the antibacterial effect of daptomycin has been observed only above this threshold concentration. The CMC value depends upon pH, temperature and bivalent metal ion concentration (Ca 2þ , Mg 2þ , Ni 2þ , Cu 2þ etc.). In our laboratory, studies of bacillomycin activity in model membranes have revealed a relationship between activity and LP micelle size, which is independent of the LP's own affinity to the respective membrane (unpublished data).

Although there are extensive studies on LP antimicrobial activity, the precise mechanisms for their membrane-peptide interactions and cell death have not been clearly established. The common mechanism described consist of a "self-promoted uptake" of the LPs to cross the cell membrane and its subsequent disruption; triggering the bacterial and/or fungal cell death [64] . Studies reveal that events associated with binding and disruption of the cytoplasmic membrane may occur through the formation of pores [65] or a detergent-like "carpet" mechanism [66] . The initial interaction of a-helical LPs with lipid bilayers could occur in any of three general orientations: parallel to the lipid bilayer, perpendicular to the membrane and at an oblique angle [67] . There is considerable evidence sustaining each of these processes, and different LPs exhibit different mechanisms to disturb the cell membrane.

Characterizing the lipid-lipopeptide interactions has been proven to be difficult. This review article is mainly focused on the analytical methods that can be used analyze and to understand the LP-lipid interaction. In the following sections, we review the most relevant methods for studying LP-lipid interactions.

2. Diffraction methods for the study of LP-lipid interactions 2.1. X-ray diffraction X-ray crystallography-Among the tools used for structural characterization, X-ray crystallography is the standard method. It enables the elucidation of the atomic structure of threedimensional LP-lipid complexes and the binding sites of LPs in Tolassin  19e25  3-hydroxydecanoic acid or 3-hydroxydodecanoic acid  2, 3-dehydro-2-amino-butyric acid and homoserine  [42]  Syringomycin  9 3-hydroxydecanoic acid or 3-hydroxydodecanoic acid 2,4-diaminobutyric acid C-terminal chlorinated threonine [43] the cell membrane. The graphic display of atomic structures reveals the binding site location and presence of the bound ligand. X-ray diffraction studies on cell membranes allow the evaluation of the structure of membrane proteins, mechanism of membrane transport proteins and the structure of ion channels (Table 3) . These aspects of X-ray diffraction applications in membrane science have been reviewed by Yigong Shi [68] . Most analytical techniques examine LPs secondary structure, as well as the localization of the individual residues on membranes. Likewise, structural changes in the membrane over the course of peptide interaction is another important criterion that needs to be elucidated. The recent development of X-ray diffraction structure and phase property of fluid membranes with short-range order of lipids in the presence of peptides allows this feature to be examined. Recent advancements in this area transformed X-ray diffraction into a powerful method to elucidate LP-lipid interactions in the hydrated state of the lipid bilayer and spontaneous insertion of LPs into membranes. There are two types of information about LP-lipid interactions that can be obtained from X-ray diffraction techniques. One is data on the position of lipids in the membrane layer (at resolutions up to several angstroms (Å), using the heavy atom labeling technique, and the other is the data given by these techniques related to membrane thickening, thinning and packing during LP-lipid interactions.

Multiple Wavelength Anomalous Diffraction (MAD)-With the development of Multiple Wavelength Anomalous Diffraction (MAD), considerable improvement has been achieved allowing to decipher the 'phase shift characteristics' in biological membranes and model systems [69, 70] . Elucidation of the lipid structure is an important aspect of LPs interaction with membranes, since most of them form pores. With the combined use of MAD and the heavy atom labeling technique, information about the lipid structure of the membrane and position of the AA residues of the LP in the membrane layers are revealed. For example, using thallium as the anomalous scattering atoms, the MAD method has been used to determine the lipid structure of membranes containing gramicidin ion channels [69] . Lately, the MAD method was successfully used to solve the lipid structure of the inverted hexagonal phase of a phospholipid with brominated chains. The bromine distribution obtained from the MAD analysis provided the details of the chain packing in the hexagonal unit cell, through the observation of the intensity undulation of the bromine distribution around the unit cell [70] . The change in the density profile of the bilayer enables the determination of the location of the LP in the bilayer. For example, White et al. developed bilayer profiling by labeling a specific double bond of the lipid. Using this method, they studied the location and orientation of the membrane-bound peptides [71] .

Vital information about membranes can be retrieved when biomimetic multilamellar lipid membranes supported on solid surfaces are irradiated by X-ray [72] . This method offers a novel and non-destructive approach to investigate the structure of lipid membranes, with and without the presence of membrane-active agents such as LPs. With the aid of modern interface sensitive Xray scattering techniques, a precise distinction can be made between the normal q z and parallel q || scattering vector component of the membrane bilayer. With this technique, the lateral structure of bilayers from weakly ordered systems can be elucidated. One factor to consider is that LP incorporation into multilamellar structures is achieved under non-spontaneous insertion.

The principal mode of action of antimicrobial LPs is through direct cell membrane interaction, rather than cell lysis. Hydrophobic matching mediated by direct interaction of LPs with cell membranes causes subsequent membrane thinning or thickening and lipid reorientation of the membrane. Hence, the X-ray scattering method should be an apt method to probe LP-lipid interactions in the fluid state of the bilayer. Scattering experiments on lipid films could possibly yield evidence in several ways. For example, the vertical density profile of bilayers r(z) (averaged in the XY plane) and the lateral bilayer irregularities through diffuse scattering, the lateral membrane structure on molecular length scale using Grazing Incidence X-ray Diffraction (GIXD) and the ordering of peptides on the surface of the membrane bilayer through Grazing Incidence Small-Angle X-ray Scattering (GISAXS).

Grazing Incidence X-ray Diffraction (GIXRD)-Studies on the molecular structure of membrane surfaces over the course of LP-lipid interactions is also very important. This can be evaluated using GIXRD [72, 73] . By means of in-plane diffraction of periodically organized lipid films, one can obtain high-resolution information about the membrane surface. In a typical GIXRD measurement an incident X-ray radiation with a 1.5 Å wavelength is set to strike at the air-water interface of the membrane at an incident angle (0.8 a c ), below the critical angle of total reflection (a c ). Otherwise, this would lead to total external reflection, having the refracted waves becoming evanescent waves. Evanescent waves travel below the surface parallel to the interface, with a typical penetration depth of 76 Å. In sufficiently long-range ordered membranes, the ordered structure of monolayers can be diffracted. In the event of LP-lipid interaction, GIXRD would allow the detection of two fundamental factors: firstly, the partial ordering of the peptides and subsequent change in intensity distribution, which can be correlated to pore size, orientation, and conformation [74] ; secondly, the measurement of area per lipid molecules before and after the introduction of the LP. Using this method, Gidalevitz and coworkers studied the interaction of lipid A (a major component in the outer membrane of gram-negative bacteria) with AMPs such as LL-37, SMAP-29 and D2A22 [75] . During a constant pressure experiment, they observed that at higher L/P ratio there was an increase in the area per lipid molecule. Similarly, a study of the interaction between the ovine AMP SMAP-29 and phospholipid monolayers using GIXRD revealed the same proportional increase in the area per lipid [76] .

According to the study of Huang and co-workers, there was a concentration dependent phase transition occurring at critical peptide-lipid ratio (P/L). They evidenced through circular dichroism [84] (CD) that, in aligned membranes, the transition of a-helical peptides from the parallel state to the inserted state caused a phase transition [77, 78] . The transition from the parallel state to the inserted state of the LP showed a sigmoidal concentration dependence. Furthermore, they established a correlation between this transition and membrane elasticity, as well as other physical parameters such as membrane thickness. During recent years, X-ray diffraction contributed greatly to these studies. First, it was determined that the location of LPs in the bilayer was directly correlated with the changes in the electron density. For example, melittin-MLM in DOPC showed a substantial increase in the PC head group due to its insertion in the bilayer [79] . Small-Angle X-ray Scattering (SAXS) and Wide-Angle X-ray Diffraction (WAXD)-Hydrophobic matching and thinning or thickening of the bilayer during interaction with LPs have been studied through Small-Angle X-ray Scattering (SAXS). In-depth analysis of scattering data has been made possible through the use of advanced software and hardware technologies [80, 81] . Due to technological advancements of the third-generation synchrotrons and X-ray detectors, there is a growing demand for SAXS in the structural biology community. Typical SAXS experiments involve recording the scattering at small angles (typically 0.1e10 ) and the elastically scattered waves of the X-ray beam impinging on electrons ( Fig. 1 ). Unlike other structural techniques, the scattering curve can always be measured without having a well-diffracting crystal, such as the one required for crystallographic analysis. Using background-subtracted SAXD, one can obtain the parameters of the d value that is the sum of membrane thickness (d B ) and thickness of water layer (d w ) [d ¼ d B þ d w ] and the 1D electron density profile calculated from SAXD diffractograms. For instance, Ortiz and coworkers studied the interaction of lichenysin with dipalmitoylphosphatidylcholine (DPPC) membranes via the SAXD method [82] . They revealed that, though the presence of the LP did not alter the lamellar structure organization, the interlamellar repeat distance increased. The electron density profile also revealed that there was an increase in the d value, due to the insertion of lichenysin in the DPPC membrane bilayer. Likewise, the interaction of an antimicrobial surfactant-like peptide containing a cationic head group (Ala) 6 (Arg), A 6 , with zwitterionic DPPC lipid vesicles was investigated by Hamely and coworkers. SAXD data revealed that the local multilamellar organization of DPPC vesicles was disturbed, possibly due to the swelling effect [83] .

Like SAXS, Wide Angle X-ray Diffraction (WAXD) is another technique giving information about the aliphatic chain lattice, and thus, the bilayer packing at the Å scale; though requiring the presence of crystalline-like ordered phases. In WAXD, the distance from the sample to the detector is shorter, thus allowing for the observation of the diffraction maxima at longer wavelengths [82, 84] .

Although the X-ray diffraction method is suitable for pure LP- Fig. 1 . Schematic representation of ideal small-angle scattering experiment (i) as a function of the parallel (q k ) and normal (q z ) components of the momentum transfer (q). (ii) in the vicinity of the (specular) q z axis. In this study, the q z components (iii) were measured by line scanning under specular conditions (iv). At low q z , the lipid acyl chain correlation maximum (v) is observed. Lorentzian fits yield the lateral lipid chain distance. The width of the acyl chain peak along qk gives information about the lipid ordering (correlation length); the angular width of the peak corresponds to the acyl chain tilt. In addition, superstructures (vi) and peptide geometries (helix maximum, vii) can be observed in some cases. Figure lipid model system analyses, it has limitations. For example, X-ray diffraction cannot be applied to study the interaction of larger LPs with lipids unless the membrane active segment of the peptide is split (or else the process becomes laborious). Additionally, X-ray diffraction can only give partial information, even for the highly ordered state of the fluid membrane, and it is not possible to quantify the results below a certain threshold. As previously mentioned, in order to get observable scattering peaks, the lipidpeptide ratio should be high. Nevertheless, summing up the results from X-ray diffraction with the one obtained from additional experimental approaches (Molecular Dynamic simulations, solidstate NMR, CD measurements on oriented membranes and other spectroscopic techniques) it is possible to get high-resolution structural information of LP-lipid interactions.

Infrared (IR) spectroscopy is a suitable method to explore LPlipid interactions at the molecular level (Table 4) . IR spectroscopy mainly reveals information about functional group (-NH, eC]O) status during LP-lipid interaction. FTIR can also report on the effect of LPs on the symmetric or asymmetric CH2 vibrations of lipid chains, highly abundant in any membrane preparations. The frequency of CH2 vibrations depends on the conformation of lipid acyl chains. With the development of the Attenuated Total Reflectance (ATR) technique, the study of LP-lipid interactions via IR spectroscopy became less complicated [108e112]. In ATIR-FTIR technique, the sample is probed by the evanescent wave produced by the total reflection of the light. For the total internal reflection, a crystal (Germanium or ZnSe) with a greater refractive index than the sample is used (Fig. 2) . The absorption of IR radiation by peptides depends on the type of chemical bonds, type of bond vibration and the mass of the atoms involved. The IR absorbance contribution of the AA Amide I bond of the peptide (stretching vibration of eC]O) usually appears in the 1600 -1700 cm À1 region [113] . The strength of the hydrogen bonding depends on the secondary structure of the peptide, and it affects the absorption frequency of the C]O vibration. Samples can be prepared by dissolving LP-lipid mixtures in 1:2 MeOH/CH 2 Cl 2 and dispersing the solution on the top of the prism [114] . Excess D 2 O hydration of the samples is achieved by incubating the lipid/peptide mixture for 2 h in the prism-chamber before spectra acquisition. By correlating the amide I frequency observed with the typical a-helical, b-sheets and random coil secondary structure spectra, the structural changes of the lipidpeptide interaction event can be elucidated. When an a-helical LP binds with the membrane, the C]O band center appears in the range of 1656e1658 cm À1 , whereas, in solution, the same band absorption appears between 1650 and 1655 cm À1 . In the case of deuterium exchanged helices, since the band frequency depends on the nuclei involved in the vibration, the Amide I band can shift to a frequency as low as 1644 cm À1 . Assignment of bands in thẽ 1660e1640 cm À1 region is complex because of the overlap of ahelical and random structures of the absorption profiles. Fortunately, this difficulty can be addressed by exchanging the Amide I hydrogen with deuterium [115] . In a random coil structure, the deuterium substitution results in a large shift in the C]O band (up to around 1646 cm À1 ). In an a-helical structure, the substitution perturbs the C]O band minimally. As the deuterium exchange depends on the accessibility of the water to Amide I and II, this procedure gives information about the LP depth of insertion in the hydrophobic membrane. For example, the determination of model b-sheet peptide, P 11-2 (CH 3 CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH 2 ) membrane interaction using infrared spectroscopy (hydrogen-deuterium exchange) revealed that the LP b-sheet structure was induced by membrane contact, forming larger aggregates of b-sheet [116] .

Understanding the structural changes occurring during the initial events of the lipid-LP interaction is a crucial research field. Lipid bilayers experiments failed to study this event. To this end, using lipid monolayers as experimental models to study lipid-LP interactions at the air-water interface presents several technical advantages [117] . First, the experiments can be done on aqueous substrates, which are biologically more relevant than solid surfaces. Second, many biophysically relevant experimental parameters (such as surface pressure, area occupied by the molecule, temperature, and pH) can be controlled. Several techniques, such as fluorescence Microscopy (FM), Brewster Angle Microscopy (BAM), and X-ray diffraction allow the characterization such as phase behavior. Nevertheless, structural information of the film organization was unavailable until the emergence of IR spectroscopy. Using the IR spectroscopy procedures described in the reports of Dluhy et al.

[118e120], it is possible to acquire structural information from monolayers formed at the air-water interface. The technique introduced is a variation of IR spectroscopy named Infrared Reflec-tioneAbsorption Spectroscopy (IRRAS). In a typical IRRAS spectroscopy, a perpendicularly polarized (s-polarized) or parallel polarized (p-polarized) IR beam is allowed to impinge on the surface at a well-defined and controlled angle of incidence (Fig. 3) . The reflected light is detected at an angle equal to the incidence angle. The interference arising from the rotation-vibration bands from water can be tackled by two methods. First, the sample shuttle method, in which the film surface and the film-free surface are reflected, and the ratio of their spectrum is extracted [121] . The second approach consists of Polarization ModulationeInfrared ReflectioneAdsorption Spectroscopy (PM-IRRAS). In this method, alternating linear states of light are generated by a photo elastic modulator, which substantially reduces the interference of water and carbon dioxide [122] . The general way of presenting IRRAS spectra is Reflectance-Absorbance (RA) vs wavenumber. The primary application of IRRAS is the study of the structural and conformational changes of lipid chains in Langmuir monolayer films. The CH 2 stretching vibrational frequencies have a tendency to decrease when the surface pressure increases [123] . Thus, the change in the CH 2 stretching vibrational frequency in function of the applied surface pressure provides knowledge about the correlation between the structural changes and the physical state of the monolayer. It is also possible to get precise information about the tilt angles in the ordered phases using the data relating to the chain conformation through theoretical formalism. IRRAS also allows one to get an insight into the structural changes in the polar region (phosphate groups) of the lipid monolayer. The phosphate group vibrational frequency is sensitive to hydration (H-bonding). The hydration state of the phosphate group (monohydrated or dehydrated) depends upon the surface pressure of the monolayer. The compiled interpretation of these techniques reveals information about the polar head group's structural and environmental changes in the vicinity of water.

With slight changes in the technical approach, IRRAS can provide information about protein conformational changes in monolayer films. For instance, IRRAS allows monitoring the temporal stability of peptide structure in films, alone and in the presence of the lipid interface over long periods of time. The vibrational Amide I region frequency shift in the presence of the lipid surface reveals the structural changes of the peptide during the interaction [124] . This method also permits the study of surface pressure-induced secondary structure change of peptide fragments. For example, Shibata and coworkers carried out a PM-IRRAS study to determine the secondary structure of the pulmonary surfactant model peptide, Hel 13e5, in the absence and the presence of phospholipid monolayers at the air-water interface [125] . Orientations of the peptide and lipid chains can be determined by PM-IRRAS. Orientation and lipid-peptide interactions of gramicidin A in lipid membranes was studied by Kota et al. [126] . They found that surface pressure increases upon compression at the interface of the phospholipid monolayer, and that the conformation of Hel 13-5 changed from a-helix to b-sheet. In summary, the FT-IR spectroscopic technique can give information about lipid/LP interaction, information on the lipid monolayer structure, secondary structure of the interacting peptides and their orientation. In addition to this, the techniques mentioned above require a minimal quantity of the sample. Although FT-IR has many advantages; some disadvantages include the lack of quantitative information and difficulty in forming monolayers with certain lipids.

Fluorescence can be defined as the emission of light by a substance that has been excited by light. The wavelength of emission is always higher than the excitation wavelength. Fluoresce intensity (I F ) and wavelength (l F ) are the simplest parameters used to monitor molecular interactions in the biological systems. Fluorescence intensity (I F ), at the given excitation wavelength (l E ) and emission wavelength (l F ), is given by equation (1).

Where k is a constant depending upon the measurement apparatus, F(l) is the total emission intensity of the sample, I 0 (l E ) is the excitation intensity and A(l E ) is the absorbance of the sample. Therefore, I F is a relative parameter and has a linear correlation with the concentration of the sample. Fluorescence intensities of molecules containing environment-sensitive fluorophores are frequently used to study molecular interactions. There are two reasons that can explain the fluorophore environment changes. One is the binding of the interacting partner in its close vicinity, and the second is the conformational change of the molecule containing the fluorophore evoked by the binding process. To determine the mechanism of LP-lipid interaction, intrinsic fluorescence of the LP can be used in several ways (Table 5 ). LPs containing tryptophan (trp) and/or tyrosine (tyr) have significant intrinsic fluorescence, which is a valuable tool for quantifying their insertion into the lipid membranes. The fluorescence intensity of these amino acids depends on the physical property of their microenvironment, such as their hydrophilic and hydrophobic nature. Hence, LP insertion into membranes leads to changes in the microenvironment of the fluorescent amino acids, which in turn can lead to substantial changes in quantum yield, the wavelength of the emission maximum, fluorescence anisotropy and fluorescence lifetime [146] . From LP fluorescence emission differences observed in the aqueous and in the membrane environment, it is possible to determine the partition constant. The partition constant (K) (eq. (2)) is defined as the ratio of the peptide molecules embedded in the lipid (L) phase, over the peptide molecules in solution in the aqueous phase (W) [147, 148] .

Where n S,i are the moles of solute present in the aqueous (i ¼ W) and lipid (i ¼ L) phases and V i is the water (i ¼ W) and (i ¼ L) lipid volume. Hence, fluorescence response of free (I W ) and bound (I L ) peptides could be used to quantify the K P using the following equation (Eq. (3)). I ¼

Evaluation of a LP partition constant K p in various lipid systems provides knowledge about its lipid specificity. For example, partition studies of Psd1, a 46 AA residue defensin isolated from seeds of the pea Pisum sativum, was carried out by Santos and coworkers through fluorescence studies [149] . Their results revealed that Psd1 had a marked selectivity towards model membranes containing ergosterol; reaching uncommonly high partition coefficient (Kp) values, while no partition was observed in the case of model membranes containing cholesterol. LP families from Bacillus subtilis strains, such as surfactins (SF), iturins (IT), and fengycins (FE) also show a similar kind of selectivity in model membranes [150] . MP-1, a peptide from the venom of the Polybia paulista wasp with selectivity to Leukemic T-Lymphocyte cell, showed smaller partition coefficients with cholesterol-containing membranes [151] .

The quenching phenomenon can be used to study the depth of aromatic amino acids (tyr and trp) present in LPs. After the interaction of the LPs with model membranes; acrylamide (a watersoluble quencher of tyrosine and tryptophan) [152] access to fluorescent amino acids is studied and their quenching Stern-Volmer constant can be calculated. Acrylamide is the most frequently used quencher for LP-lipid interaction studies. It's solubility in water and low penetration in lipid bilayer make it a suitable quencher for the biological applications [153] . Similar studies can also be carried out with brominated lipids [154, 155] . The quenching Stern-Volmer constant is the measure of binding efficiency between LP in solution and in model membranes [100] . In addition, the emission spectra of LPs undergo spectral shift (red or blue shifts) in the membrane environment, which can indicate insertion or membrane interaction.

By suitable site-directed LP labeling (with fluorophores), it is possible to study the structural behavior of LP during membrane interaction [156e161]. 7-Nitro2,1,3-benzoxadiazol (NBD), Alexa Fluor 350, perylene and pyrene are amongst the best-known fluorescent labeling agents [162, 163] . These molecules show fluorescence emission shifts when their neighboring components change (for instance, during translocation from water to an apolar and highly viscous environment) or upon oligomerization (excimer formation). Among the fluorophore labels stated above, pyrene is the most established fluorescent probe [164] . Pyrene is generally conjugated with LPs by labeling lysines or cysteines that contain reactive functional groups such as succinimidyl ester, isothiocyanate, sulfonyl chloride, maleimide and iodoacetamide [165, 166] . When LP-lipid interactions with LP-labeled pyrene are carried out, two important aspects of the spectral fluorescence should be considered:1-The Py value and the excimer emission. The Py value is defined as the ratio of the fluorescence intensity of band I and band III of the pyrene-labeled LP. The fluorescence emission spectrum of pyrene is characterized by five emission peaks at various wavelengths (~375, 379, 385, 395 and 410 nm) which are designated as bands I, II, III, IV and V. Among them, band III (385) is sensitive to hydrophobic environments and its intensity tends to increase significantly. In contrast, band I is sensitive to polar environments [167, 168] . Hence, the ratio of these two peaks gives information about the polarity of the probe's environment. 2 -The second phenomena, called excimer formation or excited state dimer formation, arises when two pyrene units are in close proximity (~10 Å). The excimer formation in pyrene-labeled LPs leads to the appearance of a broad, unstructured fluorescence band at longer wavelengths (ranging from 425 to 550 nm, but centered around 460 nm). This data can be employed to elucidate the microenvironment of the LP. The fluorescence emission band of the pyrene excimer deviates from the mirror-image rule; hence, the ratio between the fluorescence intensity of monomer and excimer (monomer/excimer (m/e) ratio) is a relative indicator of the extent [213] V4 [214] of excimer formation. Apart from monitoring steady-state fluorescence of pyrene-labeled LPs, quantitative analysis of timeresolved fluorescence can also be used to obtain detailed information about the LP microenvironment. For example, Jean and coworkers exposed the Ca 2þ dependent binding of pyrene-labeled A54145 [169] monomers with membranes through fluorescent studies. This method also demonstrated that the second transition of LP absorbance observed during Ca 2þ concentration increase was due to oligomer formation [170] .

Another biophysical use of fluorescence in the field of LP-lipid interaction is the study of the self-quenched carboxyfluorescein or calcein fluorescence increase [171] when these are encapsulated in lipid vesicles. Calcein has a tendency to self-quench at higher concentrations (>80 mmol) and the fluorescence intensity tends to increase at a lower concentration. This calcein behavior has been used to study liposomes vesicle leakage [117,172e174] . Another method is using the dye and quencher in the same vesicle. When the vesicles leak concomitant dequenching of the dye occurs [175] .

An alternative important tool for LP-lipid interaction studies is the F€ orster Resonance Energy Transfer (FRET). FRET is a photophysical process where the transfer of energy occurs from the excited fluorophore (termed donor (D)) to the acceptor chromophore (A). The FRET process takes place only when the electronic absorption spectrum of the lipid-LP overlaps with the emission spectrum of D. FRET depends on the distance between the donor and the acceptor without need for photon emission or molecular contact. The donor-acceptor distance at which the D/A pair energy transfer is 50% is defined as the F€ orster radius, R 0 . The value of R 0 usually lies between 2 and 6 Å and is characteristic for each D/A pair in the given environment. For a pair of donor and acceptor, the FRET energy transfer (E) is inversely proportional to the sixth power of the distance (eq. (4)).

Where R 0 is the F€ orster radius or F€ orster energy transfer distance, at which FRET efficiency is 50%. R 0 can be determined according to the following equation (eq. (5)) [176] .

Where, n is the refractive index of the medium in which fluorescence is measured, F D is the quantum yield of the donor, a is a constant (8.785 Â 10 À25 M cm 3 ) and J is the normalized overlap between the integral spectrum of donor emission and acceptor absorbance spectrum. J can be given by the following equation (Eq. (6)), where ε A represents molar extinction coefficient.

The vesicle binding of LPs containing fluorescent AA (donor such as trp) can be studied by the FRET technique. In the event of a LPvesicle interaction, an increase in the FRET signal is observed. Vesicles labeled with nitrobenzoxadiazole (NBD) are titrated with LP, and the relative change in the fluorescence (dF) is recorded. dF can be given by (FeF 0 )/F 0 , where F and F 0 represent the fluorescence intensity with and without the LP. Binding of the polymyxin B derived LPs, sp-34, sp-96 and sp-100, with various vesicles of distinct lipids composition labeled with NBD was studied by Yolanda and coworkers [177] . Activity of the LPs and their anionic phospholipid selectivity were elucidated through monitoring the FRET fluorescence (dF). Most of the LPs membrane interaction mechanisms are accompanied by the formation of oligomers. The FRET method is a useful analytical technique to monitor LP oligomer formation. FRET has been used to understand daptomycin oligomerization and its effect on model membranes [178e183] .

Fluorescence Correlation Spectroscopy (FCS) is another fluorescence technique variation that can be used to study the interaction of AMPs with membranes. FCS is a noninvasive procedure that can be used to investigate the interaction of labeled molecules. This method involves monitoring fluorescence intensity fluctuations of labeled particles contained in a small confined volume. The variations are caused by deviations from thermal equilibrium. The theory and the applications of the FCS technique have been discussed in many reviews [184e188] . The biological function of molecules is not only based on structure, but also on their mobility and dynamics, and FCS can be used to explore both parameters. These properties are strongly influenced by the environment. By averaging the passage of a large number of single molecules in the measurement volume, it is possible to assess their molecular movements. In principle, FCS allows the measurement of the autocorrelation function, G(t), which is a measure of self-similarity of the signal after a lag time (t). It can be described as the probability of finding a fluorescent particle at a later time, t, given that it was there at t ¼ 0. Hence, this function gives information about particle concentration or diffusion coefficients, which in turn allow the determination of molecular binding and aggregation. Fluorescence intensity fluctuations (F(t)) are detected exciting the volume with a single photon. At equilibrium, the auto-correlation function G(t), which is a measure of similar fluorescence signals over the period of measurement in a given volume, can be defined by the following equation (Eq. (7) ). 

In order to get the detectable intensity of fluctuations, the average number of particles residing in the excitation volume should be low. Pramanik and coworkers [189] applied the FCS technique to study the interaction between Alzheimer amyloid bpeptide (Ab) and cultured human cerebral cortical neuron cells. Through FCS measurement of rhodamine-labeled Ab (Rh-Ab), they found three diffusion times: 0.1, 1.1 and 5.9 ms. 0.1 ms diffusion time corresponded to the unbound Rh-Ab, and 1.1 and 5.9 ms corresponded to slowly diffusing complexes of Rh-Ab bound to the human cerebral cortical neuron cells.

Since FCS involves the measurement of an average number of fluorescent particles diffusing in a given volume, it can also provide a ratio between average fluorescence intensity versus an average number of fluorescent particles. In this way, a simple comparison of the average brightness per particle with the brightness of the monomeric form gives information about the oligomer state of the molecule. Hence, FCS can be used to study membrane-induced aggregation of LPs. For example, Nag and coworkers [190] studied the aggregation formation in amyloid-b (Ab) LPs on membranes of living cells. At a concentration of 350 nM, they found a higher population of larger aggregates than at a lower concentration (150 nM). They also found that the membrane environment was necessary for the oligomer formation, even when the concentration of amyloid-b (Ab) was high.

Circular dichroism (CD) is a rapid analytical method that can reveal the structure of membrane-bound LPs. Recent advances are summarized in Table 6 . CD involves the measurement of chiral molecules differential absorption of right and left circularly polarized light. When circularly polarized light passes through the sample; both components of light (right and left circularly polarized light) are absorbed to a different extent, depending on the chiral molecule. The resulting elliptical polarization (CD effect) is measured by spectropolarimeter. The far-UV CD (260e180 nm) spectra, which originate from the peptide light absorption, can give information about the secondary structure of LPs. The near-UV CD spectra (320e250 nm) results from the absorption of AA residues (trp and tyr), and depends on the tertiary and quaternary structure of the peptide. The secondary structural features of oligopeptides, such as propensity to form a-helices in solution and aggregation formation (by aligning b-sheet structures) have been studied by far-UV CD techniques [215, 216] . A typical CD spectropolarimeter is equipped with a multi-position Peltier-controlled cell holder, a Xenon lamp and a temperature controller. The CD instrument can be purged with nitrogen gas in order to maintain an inert atmosphere during the experiments. The main advantages of the CD technique include the relatively low amounts of samples required and the fact that no peptide labeling is necessary.

AMPs disintegrate cells by perturbing their outer membrane. CD studies carried on model membranes have allowed the determination of peptide structural change in the membrane environment [217e219]. For example, Huang and coworkers [220] studied the molecular state of membrane active antibiotic daptomycin through detailed CD spectral analysis. They found the existence of two different states of daptomycin in the presence of Ca 2þ and PG containing membranes: before and after membrane binding. All other CD spectral signals were intermediate states of these two states. The molecular state of the daptomycin was similar, irrespective of Ca 2þ presence, and change in the molecular state was observed only after membrane binding. Romanelli and coworkers [221] explored the secondary structure of two AMPs: magainin 2 and cecropin A, with E. coli bacterial cells, through CD analysis. They reported that the secondary structure change of these AMPs (magainin 2 and cecropin A) was driven by the outer membrane components of E. coli bacterial cells.

Though there are numerous CD studies on peptide-membrane interactions, the conventional CD analysis method fails to yield any information about the orientation of the peptide relative to the membrane. When the binding affinity between the peptide and the lipid bilayer is low, the CD signal from the peptide is also very low. The development of Oriented Circular Dichroism (OCD) addresses this issue. The advantages of the OCD technique are that (i) very little sample quantity is needed for analysis (typically 3e10 mg of peptide per sample), (ii) it does not require peptide labeling and (iii) OCD can reveal structural perturbations caused by NMR-/ESRlabels or other mutations [222] . OCD structure analysis of a-helical peptides is based on Moffit's theory, which can predict the dipole moment (m) of p-p* transition of the amide bond. These are polarized parallel or perpendicular to the helical axis. The OCD spectrum of helical peptide bound to the membrane ordered plane gives a specific trace shape (Fig. 4) . The intensity of the OCD line bands depends on the orientation of the peptide. The intensity of the negative band around 208 nm depends on the helix orientation, and its presence or absence is indicative of surface or transmembrane helix alignment. From the results of numerous lipid-LP interaction studies using different analytical techniques, it became evident that peptides show a drastic change in their alignment in response to environment and/or experimental conditions. In this regard, the OCD spectra of peptides readily show whether they are bound to the membrane surface in one of the following states: "Sstate", inserted "I-state" or bound in tilted "T-state".

Numerous OCD analyses have been carried out on various AMPs, such as alamethicin [223e226], melittin [227, 228] and magainin [229] in the membrane environment. All these studies reveal that peptides are able to change their orientation in the membrane. At lower P/L ratios, OCD spectra have shown the prevalence of helix orientation parallel to the membrane surface (in the S-state). Above a specific P/L ratio, the peptides change their alignment to helix conformations perpendicular to the membrane (the inserted Istate). The intermediate state of the peptide-membrane bilayer interaction is a linear combination of S-state and I-state. The helix alignment can be influenced by peptide concentration, the hydration state and lipid composition of the membrane. Each of these conditions create distinct OCD spectra that can be studied reversibly and continuously [230, 231] . The alignment of the anionic amphiphilic peptide Dermicidin (DCD-1L) on different phospholipid membranes was studied through OCD by Schutte and coworkers [232] . DCD-1L showed the most pronounced a-helical structure on POPG membranes at a P/L ratio of 1:50 (mol/mol). The distinct OCD negative band observed at 208 nm showed that DCD-1L was aligned parallel to the membrane surface. When DCD-1Lwas in contact with a membrane made up of DMPC/DMPG (1:1 mol/ mol) at the same P/L, a similar OCD spectral behavior was observed, though with less intensity. The loss of spectral intensity was attributed to DCD-1L agglomeration induced by DMPC/DMPG membranes. From the normalized OCD spectra, they concluded that the alignment of DCD-1L on POPG and DMPC/DMPG was the same, irrespective of the oligomer formation. In a similar approach, Straus and coworkers [233] studied the effects of the AMPs from the Australian southern bell frog, Aurein 2.2 and 2.3, on membranes of various lipid compositions. In membranes of three different lipid compositions, the OCD bands showed that all three peptides were surface adsorbed at a low P/L ratio. The 'I' state was observed at a higher P/L ratio.

Analyzing pH-triggered transmembranal helical structure is also possible by OCD. Investigations of pH-dependent membraneassociated folding have practical applications for targeting acidic tumor cells and drug delivery across cancer cell membranes [234] . Reshetnyak and coworkers [235] studied interaction of the truncated version pH (Low) Insertion Peptides (pHLIP® peptides) with lipid bilayer membranes. They identified that the truncated version of pHLIP® peptides was dependent on interaction and helix formation with membranes. The detailed OCD applications for analyzing peptide interactions with membranes were reviewed by Ulrich and coworker [236] .

Nuclear Magnetic Resonance (NMR) spectroscopy is a unique biophysical method that can be used to study various aspects of ligand-receptor binding. NMR can be used to identify the potential binding ligand (who), to identify the binding site of the ligand on the macromolecular receptor (where) and to identify the binding mechanism (why). The development of several NMR techniques, such as the isotopic labeling method, paramagnetic magnetization or magnetic transfer, enabled detailed elucidation of the molecular recognition process; substantially more complex than the original lock-and-key model. Though NMR experiments using isotopic labeling of ligands or receptors can yield valuable information, NMR is mainly a label-free detection method. From a chemical point of view, the radioisotopically labeled molecules and their native form have indistinguishable chemical properties. When the compound is soluble, NMR provides sharp signals with good resolution and unambiguous detection of individual compounds. Furthermore, its sensitivity is very high, even for weak binding interactions. NMR can determine affinities with ligand concentrations well below the K d . Albeit NMR has many advantages, it is not without drawbacks. When the energy levels of the NMR transitions are very close, signal intensity and mass sensitivity are low.

The basic principle of NMR spectroscopy involves the interaction between the magnetic moments of some atomic nuclei and an applied external magnetic field. The energy difference between the spin states resulting from the applied magnetic field depends on the nature of the nucleus, the magnetogyric ratio g and the magnetic induction B 0 . The energy difference between spin states are very low and fall in the radiofrequency range (hundreds of MHz to GHz). Energy (E) can be given by the following equation (Eq. (8)).

Where ħ is the Planck constant divided by 2p. NMR can detect the frequency shifts resulting from subtle changes in the electronic environment of the nucleus. This feature makes NMR a suitable candidate to study the specific sites of structural changes in peptide-lipid interactions; as the specific resonance line from each hydrogen nucleus depends on its electronic environment. A nucleus can be detected by NMR only when its spin quantum number is above zero, preferentially ½. For example, 1 H, 19 F, 31 P, and 13 C are some of the nuclei observable by NMR. Besides resonance frequency, there are other parameters that can be obtained from the NMR method. First, the integral of the resonance peak is proportional to the concentration of the compound. Second, nuclei interactions through bonds lead to spin-spin interactions, allowing to discriminate various protons present in the compound. Third, the relaxation process after perturbations of the nuclei provide information of the dynamics and the binding event of the compound. Through NMR it is possible to study certain parameters of LPlipid interactions ( Table 7 ). The molecular weight range of the peptide and its complex with lipids should fall in the permissible weight range (~30 kDa) of NMR, in order to be studied by highresolution NMR approaches. The overall fold is the characteristic feature of the LP. In addition, the NMR technique can also reveal the mechanism of interaction of LPs with the chiral receptors, such as, lipid II. Detailed NMR studies has been carried out for LPs such as Polymyxin B, Nisin and its interaction with chiral receptors [254e257]. Solution-state NMR can give information about the presence of loops or loose termini in the folded structure of LPs. Proper selection of the membrane bilayer model is vital to investigate lipid-peptide interaction through liquid-state NMR [258e262]. The structure, location and dynamics of the peptide on the membrane depend on the choice of the membrane mimetic. In addition, for a good resolution spectrum, the reorientation of the membrane is a strict requirement. Typical membrane models for biophysical studies of LP-lipid interactions can be classified as vesicles [263e265], bicelles [266, 267] and detergent micelles [268] . Vesicles can be produced by a large variety of phospholipids with a variety of head groups (neutral, anionic or cationic), acyl chain length, unsaturation and with the incorporation of sterols (cholesterol/ergosterol). However, due to their large size (LUV) or very large curvature (SUV), vesicles are far from being the ideal candidates for NMR. For instance, Cuccovia and co-workers [269] studied the interaction of a cecropin-melittin hybrid AMP(BP100) on negatively charged membranes through NMR. The NMR spectra showed that upon binding to PG-containing LUVs, BP100 acquired an a-helical conformation, spanning residues in the range of 3e11.

Furthermore, 15 NMR data revealed that BP100 was located on the membrane surface with the helix axis parallel to it. Almeida and coworkers [270] used NMR analysis to study the conformation and dynamics of rPsd1 in the presence of vesicles containing phosphatidylcholine (PC). The chemical perturbation and relaxation process revealed the loops as the binding site of Psd1 on the membrane surface (see Table 8 ).

For the liquid-state study of LP-lipid interaction, micellar membrane models are preferred for several reasons. The size of the micelles is small, which means their dynamics (rotation) match with the time scale required for NMR. Examples of detergents used in this kind of studies include dihexanoyl phosphatidylcholine (DHCP), dodecylphosphocholine (DPC) and sodium dodecyl sulfate (SDS) [271e273]. Unfortunately, many peptides are not active with micelles and in many cases, the micelle-peptide structure is different in other models. Another choice of membrane mimetic for solution-state NMR studies is bicelles. Bicelles are a mixture of various phospholipids and suitable detergents [274, 275] . The size and phase properties of bicelles can be adjusted using varying amounts lipids and detergents. There are many solution-state NMR investigations on small (q 0.5) isotropic bicelles that have studied the structure and membrane interactions of several LPs [276, 277] . Since the tumbling of bicelles is isotropic and it allows fast reorientation of lipids, they are suitable candidates for solutionstate NMR spectra. Moreover, sterols can also be incorporated into bicelles and its influence on the lipid-peptide interaction can be studied [278] . Recently, nano discs have become an alternative for solution and solid-state NMR (SSNMR). Nano discs are lipid aggregates of 100e200 lipid molecules, surrounded by a membrane scaffold protein or amphipathic helical peptides, typically derived from apolipoprotein A-1 [279e281]. The size of the nanodiscs (10 nm) makes them an appropriate candidate for liquid-state NMR. As with bicelles, the size, acyl chain length, the charge of the head group and sterol content can be varied in nano discs.

Solution-state NMR studies for Lipid-LP interaction studies -Solution-state NMR offers a set of well-established multidimensional techniques to determine the structure of the peptide in the free and membrane-bound state. Simple two dimensional 1 H is enough to assign the structure of small peptide systems (<10 kDa). For example, Correlation spectroscopy (COSY) [286] and Total Correlated Spectroscopy (TOCSY) [287, 288] can be used to assign the position of the backbone and side chain of the peptide, using correlating proton signals connected by J coupling. Nuclear Overhauser Effect Spectroscopy (NOESY) spectra can give data about the distance between protons in space. From these angular and distance constraints, the peptide can be identified without labeling it. Sometimes 13 C and/or 15 N peptide labeling is necessary to resolve overlapping signals. Though lipid vesicles offer a suitable cell membrane environment for peptide interaction, they are too large to be observed in NMR. This issue can be addressed by the Transferred Nuclear Overhauser Effect (TRNOE), which is feasible when the dynamic time-scale of peptide binding and unbinding gives rise to transferred nuclear overhauser signals [288, 289] . These NMR spectra are recorded from peptides in the free and vesicle-bound state. When fast exchange occurs in lipid-bound and free states, there would be a NOE transfer from a lipid to the peptide. Interaction of dengue virus fusion peptides with anionic POPC/POPG vesicles and DPC micelles were studied by the TRNOE-NMR method [290] . That study revealed that the NOE cross-peaks were due to the hydrophobic part of the membrane, corroborating the DPC bound state of the peptide. Unfortunately, the TRNOE-NMR method is not applicable to peptides which are strongly bound to membranes. To study tightly bound peptide exchange, a "trapping method" was developed. In this method, membrane-bound peptides are exposed to 1 H 2 Oe 2 H 2 O exchange for a specific period of time. Then, the exchange is quenched with deuterated methanol. The remaining protons which are not exposed to 2 H 2 O can be detected by solution-state 1 H NMR. The interaction of melittin with membranes has been studied by this method. The periodicity of the exchange rate revealed that one side of the peptide was clearly shielded by the membrane, thus revealing the azimuthal rotation of the helix [291] . Recently, the interaction of maximin-4 (27-residue cationic AMP) with two membrane mimetic environments such as SDS and 50% methanol was studied through the NOE method [292] . A significant difference in the peptide structure was observed between SDS and methanol. The NOE of the peptide in SDS revealed several helix-helix interactions and a kinked structure, but no such features were observed in methanol.

Residual Dipolar Coupling (RDC) is another solution-based NMR technique which can be used to study the tilt and the azimuthal angle of peptides in solution and in the membrane environment [293, 294] . The RDC constant depends on the nature, distance, and angle of an internuclear vector in correlation to a molecular reference frame. In order to get long-range information of peptide orientation, 1 [298] investigated the interaction of the pentapeptide leucine-enkephalin (link) in a hydrated DMPC bilayer through the RDC-NMR method. They found that the "link" molecule was flexible, switching between specific bent conformations.

An alternative solution-state NMR based approach to obtain information about orientation and localization of peptides in the [372] membrane is by tagging a paramagnetic probe in the peptide itself. When a peptide binds to a paramagnetized micelle [299] its relaxation rates are enhanced by 1/r 6 , with r being the distance between the paramagnetic center and the observed nucleus. The interior of the micelle can be paramagnetized through the incorporation of 5-, 12-or 16-doxylstearate into DPC micelles. As the micelles forming detergents and lipids are flexible in a membrane environment, the precise position of the paramagnetic center cannot be defined. Hence, paramagnetic relaxation processes can only give qualitative information about the peptide orientation. Quantitative information about the orientation or location of the peptide can be obtained determining the depth-dependent oxygen partitioning in hydrophobic environments [300e303] . In this method, oxygen is applied to micelles at a partial pressure of 20e100 atm. As oxygen has a different level of solubility across micelles, a paramagnetic gradient is generated. This gradient generates increased relaxation in the micelle and an increased chemical shift perturbation of 19 F (5 ppm) and 13 C (0.1 ppm) can be observed. This approach was used to study diacylglycerol kinase (DAGK) peptide [292] . It is also possible to add a paramagnetic compound to the solution surrounding the micelle [304e306]. In this case, the relaxation enhancement affects spins close to the surface of the micelle. The water-soluble chelate complexes of gadolinium are paramagnetic species commonly used. Unlike other paramagnetic species (TEMPO, Mn 2þ, and Ni 2þ ), gadoliniumdiethylenetriamine pentaacetic acid-diethylamideGd (DTPA-BMA) is an inert compound in aqueous solutions. The paramagnetic relaxation effect (PRE) obtained by this method for a flat surface, and, within a good approximation, for large spherical systems, depends on 1/d 3 ; where d is the immersion depth of the peptide [291] . Zangger and coworkers [307] monitored the proton longitudinal relaxation rates upon addition of the freely water-soluble and the inert paramagnetic probe Gd (DTPA-BMA) of the a-helical peptide CM15, and determined the orientation of the fifteenresidue peptide in DPC micelles. This method has also been applied to study complete positioning of the AMPs CM15 and maximin H6 in DPC and SDS micelles [308, 309] . This technique has the considerable advantage that isotopic labeling or chemical modifications of the peptide are not necessary. Solid-state (SS) NMR studies for LPs interaction studies -In solution-state NMR spectroscopy, the isotropic values for dipoledipole coupling, chemical shift anisotropy and quadrupolar coupling interactions are zero, thus, these interactions cannot be observed. In solid-state NMR spectroscopy, the signal overlap of anisotropic interactions leads to broad peaks that impede the structure resolution. In order to get well-resolved spectra, two different approaches have been introduced. The first approach is the Magic Angle Spinning determination (MAS), where anisotropic interactions are averaged by the fast spinning of the sample around the magic angle. The second approach is the uniaxial alignment of the sample relative to the magnetic field direction. This approach gives a spectrum with sharp peaks, similar to solution NMR spectra. By using various MAS solid-state NMR, different events of the peptide-lipid contact and AMP accessibility to water during its interaction with the membrane have been characterized [310, 311] .

Another solid-state NMR approach is to register NMR data using uniaxially oriented membranes. Anisotropic chemical shifts of ahelical peptides depend on the tilt and rotational pitch angle of the peptide. Hence, the anisotropic chemical shift from samples can give information about the orientation of the peptide; as NMR measurements exhibit strong dependence with their alignment relative to the magnetic field. This information enables us to study the structure and topology of membrane-oriented peptides.

Various AMPs, such as gramicidin [312] , magainins [313] and related peptides, have been investigated using this method [314e317] . Detailed knowledge about interactions of nuclei and their angular correlations are required to perform this study. The interaction tensors of several biologically important nuclei ( 15 N for peptide bond) have been studied [318, 319] . Structures and topology of 15 N-labeled Lactophoricin peptides (LPcin-I and LPcin-II) were studied by 1D solid-state NMR [320] . 15 N NMR investigations of magainin 2 and melittin on aligned bilayers revealed that the peptides were oriented perpendicular to the membrane bilayer [321] .

The most common nucleus sensitive to NMR used in the study of lipids is 31 P. Cell membranes contain a very high proportion of phospholipids carrying a phosphate moiety in their polar head group. 31 P exhibits characteristic line shapes in each of the different lipid phases. Hence, data obtained from 31 P labeling is crucial when studying the non-lamellar phases induced by peptide interaction with membranes. In the case of strong lipid-peptide interactions, the changes in Chemical Shift Anisotropy (CSA) width can be detected; revealing the structural and dynamic change of the membrane [322e324]. With MAS experiments, differences in an isotropic chemical shift can be resolved, which can be useful to study lipid mixtures. Furthermore, the affinity of peptides with different membrane head groups and lipid acyl chain lengths can be monitored. Vosegaard and coworkers [325] researched membrane perturbations and disruption by alamethicin and novicidin using 31 P SS NMR spectroscopy. They observed that the membrane remained in a planar conformation and lipids were involved in peptide anchoring. 31 P NMR relaxation of membrane-active peptides like KALP21 and dynorphin B on bicelles containing either DMPC or a mixture of DMPC and DMPG, and (DHPC) was studied by M€ aler and coworkers [326] . A comparison of 31 P values for the lipids with and without peptide showed that dynorphin B had a greater effect on lipid head group dynamics than KALP21.

Deuterium is the most versatile nucleus when studying lipids in model membranes. The deuterium nucleus has a quadrupole moment with a spin value of one. Quadrupolar solid-state NMR spectra of membrane containing deuterated phospholipids can provide information about changes in the lipid packing. Deuteration is usually done either at the phospholipid head group or along the entire acyl chain. Conformational adjustment of the lipid zwitterionic head group is induced when peptides of a certain charge interact with membrane surface [327] . This causes the directional change in quadrupole splitting. This quadrupole splitting has a direct correlation with the motional order parameter of the hydrophobic region of the membrane. Using deuterium relaxation times, one can study the local dynamic changes induced by peptides in a specifically labeled membrane region. Recently, the interaction of a number of peptides with phospholipid membranes [425] has been studied through 2 H solid-state NMR [328e330]. For example, the 2 H NMR analysis of the short multifunctional peptide BP100 membrane interactions revealed considerable membrane thinning and local destabilization upon peptide contact [331] . The 2 H-SS NMR study of the histidine-rich designer peptide LAH4-L1 on model membranes revealed that the liposomes retained their bilayer macroscopic phase even at the highest peptide concentrations investigated [332] . Fluorine ( 19 F) is another NMR sensitive nucleus (spin-1/2 nucleus) that can be substituted for another atom (usually 1 H); even though the exchange might perturb the lipid-peptide interaction. 19 F-solid-state NMR is particularly useful to study peptides at low concentrations and to observe the effect of AMPs on membranes [333e336]. In order to study 19 F-solid-state NMR, the AMPs must be labeled with a 19 F reporter group such as 4F-phenylglycine (4F-Phg), 4-CF-phenylglycine (CF 3 -Phg) or 3F-alanine (F-Ala) [337, 338] . Recently, Hechinger and co-workers [339] carried out 19 F MAS NMR investigation of 19 F labeled alamethicin and its aggregation on POPC membranes.

Polarization Inversion with Spin change at the Magic Angle (PISEMA) is one of the solid-state NMR methods which correlate 1 He 15 N dipolar coupling. This method can provide precise information about the 15 N chemical shifts occurring when peptides associate with membranes, as well as heteronuclear dipolar coupling information. PISEMA is based on polar inversion and Spin Exchange at the Magic Angle (SEMA) between the dipolar coupled heteronuclear spin. The SEMA pulse sequence suppresses the homonuclear ( 1 He 1 H) dipolar coupling by spin locking 1 H signals along the magic angle. The PISEMA spectra of the 15 N label in highly oriented a-helical peptides often give a wheel-like pattern, termed PISA (Polarity Index Slant Angle) wheels [340, 341] . This information can be used to determine the secondary structure and topology of aligned peptides [342] .

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a rapidly expanding powerful biophysical technique used to study the structural and dynamic properties of AMPs in their native environment. EPR spectroscopy measures the absorption of microwave radiation corresponding to the energy splitting of an unpaired electron when it is placed in a strong magnetic field. EPR shares the basic quantum mechanical description with NMR, but with the detection of complex interactions of nuclear spin isotopes ( 1 H, 2 H, 13 C, 14 N, 15 N, 31 P etc.) with unpaired electron of spin labels. Therefore, the sample should contain unpaired electron spin to be EPR active. One or few spin label(s) can be attached covalently to lipids or AMPs to study LP-lipid interactions. Detailed discussion of different labeling techniques for EPR analysis was reviewed by Bordignon and Bleicken [357] . EPR spectroscopy is advantageous when compared to NMR spectroscopy in terms of sensitivity. It offers up to three orders of magnitude higher sensitivity than that of NMR and does not rely on expensive isotopic labels.

Spin-label EPR can directly yield the information about stoichiometry of AMP-associated lipid, a quantity that is related to the intramembranous structure and assembly of integral AMPs [358, 359] . This method can also be extended to investigate penetration of peptides into the membrane. In addition, EPR spectroscopy can answer pertinent questions about the dynamics of both solution and membrane bound AMPs, that are indeed difficult to solve by traditional methods. In the event of LP-lipid interaction, continuous wave EPR (CW-EPR) spectroscopy of spin-labeled peptides could yield information about motion of the spin labeled side chain, solvent accessibility of the AMP and distance between two spin labels [360] . The line shape analysis of the EPR data for spin-labeled AMPs can probe structural details with spatial resolution at the backbone level [361e363]. The motion of the spin label can reflect the environment surrounding it. Changes in the spin-label mobility can yield details about the peptide-membrane binding activities. In the aqueous phase, a spin-labeled peptide or small protein rapidly tumbling, leads to an isotropic spectrum with a rotational correlation time of less than 1 ns. However, in the membrane environment, spin labeled peptides experience restricted mobility, resulting in a broader EPR spectrum with two motional components resulting from the superposition of the signals arising from a free and bound peptide [364e366]. A recent example of using EPR spectroscopy is the study of myxinidin and its mutant WMR with lipid bilayers mimicking the membranes of Psuedomonas aeruginosa and Escherichia coli [367] . While EPR has several advantages, the requirement of unpaired electrons in the probe might not be relevant for all the systems to be studied. Some of the EPR experiments need temperatures as low as 4 K and may be an expensive constraint.

Atomic Force Microscopy (AFM) is a surface probing microscopy technique. With AFM, the structure of biological samples such as cells and membranes can be explored in real time with a fraction of nanometer-scale resolution ( Table 9 ). The basic idea of AFM is to allow a local probe (usually an oxide-sharpened microfabricated Si 3 N 4 tip also called the cantilever) to interact with the experimental surface and measure the interaction force between the tip and the sample surface (Fig. 5) . A complete review of the technical aspect of AFM was written by Dufrêne [373] . Different AFM imaging modes are available, which mainly differ in the way the tip moves over the sample surface. These techniques enable the visualization of the surface structure and lateral organization, and to study inter/ intra molecular forces [374e376]. For example, in contact mode AFM, the tip is scanned over the sample while the force applied to the sample is kept constant using feedback control. Whereas in the dynamic mode, an oscillating tip is scanned over the sample surface.

Supported Lipid Bilayers (SLBs) are the biomimetic model systems used to study the property of membranes through AFM. The most common method to form SLB is the Langmuir-Blodgett (LB) method. The LB method is based on compression of lipids on an aqueous phase using moveable barriers of a Langmuir trough made of teflon. This method allows the transfer of a monolayer of amphiphilic molecules to a solid support, generally mica. The second layer is then deposited by dipping the substrate, coated with a monolayer, through the air-water interface again, from the air into the aqueous phase [377e379]. SLBs can also be prepared by fusing lipid vesicles on solid supports. In this method, suspension of unilamellar vesicles (SUVs) are supported on cleaved mica for 45e60 min at 45e60 C (in function of Tm of the membrane lipid composition), then cooled to room temperature and rinsed with the appropriate image buffer, before taking the measurement. Another notable method of SLB preparation is the hydration of spin-coated films [380e382]. This method involves the hydration of suitably prepared dry films of lipids with the image buffer. However, this method usually forms stacked bilayer with the number of bilayers ranging between 2 and 30 [381] .

In the last decade, AFM has become a well-established technique to study the nanoscale organization of phase segregation in SLBs. So far, many investigations have been carried out to study the phase properties of binary mixtures and ternary mixtures [383e387]. Recently, Zhang and co-workers [388] studied the interfacial behavior of a binary monolayer of hexadecanol/DPPE at the air-water interface through AFM. They observed a phaseseparated monolayer and determined the interactions between hexadecanol and DPPE. Increasing the content of hexadecanol in DPPE monolayer affected the lateral organization of membrane and improved its surface tension kinetics. Other important aspects of lipid bilayers such as structure and kinetics of ripple phases [389, 390] , nucleation and growth of laterally segregated domains in SLBs [391, 392] can also be addressed with AFM.

AFM is increasingly being used to study events of membranepeptide interactions, such as membrane fusion and membrane lysis. The membrane disrupting properties of several AMPs have been studied through AFM [393e397]. Using AFM, Miller, and coworkers [398] studied the mechanism of action of the Smp 24 peptide, purified from the venom of the North African scorpion Scorpio mauruspalmatus, on prototypical synthetic prokaryotic (DOPG: DOPC) and eukaryotic (DOPE: DOPC) membranes. AFM force spectroscopy of DOPG: DOPC membranes with concentrations of Smp 24 peptide between 0.4 and 1.25 mM revealed pore formation. The pore diameters ranged between 20 and 150 nm and their depths were typically 2e4 nm. When using DOPE: DOPC membranes, no pore formation was observed; instead the removal of the bilayer in stratified lines was seen. The AFM can be used to analyze the effect of AMPs on live cells. Saprobicco-workers [399] have used AFM to study the effect of the AMP Caerin on live bacteria. They established that the AMP Caerin 1.1 caused localized defects in the cell walls of Klebsiella pneumonia cells and established the pore forming mechanism of the peptide. The AFM technique can also address membrane selectivity of AMPs [400] . GL13K is a short (13 amino acid) AMP derived from the parotid secretory protein and is highly selective towards anionic lipid membranes. The AFM imaging of negatively charged DOPG with GL13K revealed the lipid structure of the disrupted membrane. The peptide ordering formed 1 nme2 nm deep holes through the membrane surface. In the case of DOPC bilayers, membrane thinning was not observed, but strands (approximately 1 nm high) were observed on the surface [401] . The membrane disrupting ability of many designed peptide models has been studied by AFM [402, 403] . Several virus derived fusogenic peptides have also been investigated by the AFM technique [404] . These peptides are short tilted segments (10e20 AA residues) with a hydrophobicity gradient oriented along the helical axis. Since their hydrophobicity increases from one end of the helix to the other, they insert at an angle of 30e60 in lipid membranes. The AFM study of a tilted peptide derived from the Simian Immunodeficiency Virus (SIV) [405] and a fusion peptide derived from the Human Immunodeficiency Virus (HIV) [406] revealed that they form stable holes in the lipid bilayer. Several peptides and proteins can form amyloid fibrils. Amyloid insoluble fibrils are assembled from soluble peptides or proteins. Amyloid fibril formation can accompany disease and each disease is characterized by the specific protein or peptide that aggregates. For example, the fibril formation of amyloid-b (Ab), human amylin (hA) and the prion protein (PrP) are related to Alzheimer's disease, Diabetes type 2 and the spongi form encephalopathies. In this regard, AFM enables the study of the formation mechanism of amyloid fibrils and their membrane association [407e411]. Recently, Leonenko and co-workers [412] observed that Ab 1e42 fibril interaction with healthy and diseased states of the neuronal membrane was different. Using AFM, they demonstrated that the interaction of Ab 1e42 fibrils was influenced by differences in topographical nano heterogeneity and the electrical surface potential of healthy and diseased states of the neuronal membrane. Frequency-Modulation Atomic Force Microscopy (FM-AFM) was used to study the morphology and growth of the amyloid fibrils formed by MinE. This revealed that amyloid-like fibrils formed by the N-terminal domain of MinE undergo further changes on an artificial membrane surface, without causing detectable membrane damage [413] .

Ever since it was first used in the early 1940s, Transmission Electron Microscopy (TEM) has continued to be an important technology in cell biology and updates are constant. TEM enables the examiner to visualize structural perturbation of the sample in nanometer scale. The TEM imaging of thin sections of plasticembedded cells can be achieved by bombarding the sample with an electron beam. The electron beams are absorbed and scattered by the sample producing a high-resolution image. Since the wavelength of electron beams are short (100,000-fold shorter than visible light photons), sub-nanometer resolution can be achieved. Sample preparation is a crucial step in TEM analyses of cells. Various sample preparation methods, including (ultra) thin sectioning, metal shadowing, negative staining, cryo-negative staining, unstained vitrified cryo-specimens and site-specific labeling of macromolecules have been developed so far. The detailed review of sample preparation for TEM is discussed elsewhere [426] .

As previously discussed, the interaction of AMPs induces morphological and functional changes on the cell membrane. Using TEM, one can visualize the cell membrane morphological changes caused by peptides (Table 10) . Recently, a number of membranes perturbing peptides have been studied through TEM [427e430]. It has been reported that Fusobacterium nucleatum can resist human neutrophil peptide (HNP)-1 antibacterial activity through different cellular adaptations, decreasing its membrane permeability, increasing its proliferation or forming biofilms. Gürsoy and coworkers [431] evaluated the morphological and functional adaptations of F. nucleatum in the presence of (HNP)-1, using TEM. TEM observation of F. nucleatum strains revealed that intracellular granules, cytoplasmatic spaces and surface roughness start to appear with increasing HNP-1 concentration. In another study, the TEM analysis of the antimicrobial effects produced by the twopeptide bacteriocin PLNC8 ab from Lactobacillus species on the membrane of Porphyromonas gingivalis revealed severe perturbations [432] . TEM images of the periodontopathogenic bacterial cells treated with this peptide showed plasma membrane rupture, which explained the leakage of the intracellular content. Recently, Sarojini and co-workers [433] investigated the interaction of Tyrocidine A (Tyrc A) analogs bearing a planar d-Phe-2-Abz turn motif with gram-negative strains. Analysis by TEM revealed the membrane rupture behaviors by the Tyrc A synthesis analogs.

In recent years, cryo-Transmission Electron Microscopy (cryo-TEM) has become a powerful method to study nanostructured liquids. This method involves ultra-fast cooling and conversion of a liquid sample to a vitrified (glassy) specimen which is then examined in TEM. This method can give a wealth of information about structures presenting different morphologies, sizes or complexity. The primary difference between conventional TEM and cryo-TEM is in the sample preparation for analysis. In the conventional chemical fixation method, the sample is adsorbed onto a carbon support film and then fixed by adding a chemical, typically a heavy metal salt solution; most commonly uranyl acetate or molybdenum salts [434] . Afterwards, the sample is air dried and analyzed with TEM. Though this method is easy to perform and inexpensive, the phase and structure of the sample tend to transform during evaporation and/or changes in temperature. In contrast, cryo-TEM fixation of the sample is made by ultrafast cooling, implying a quasiinstantaneous transition from liquid to solid. This method does not require the addition of foreign compounds and has minimal influence on the bulk conditions, composition or structure of the sample. In order to sustain the original nanostructure of the sample, the freezing procedure requires cooling rates of hundreds of degrees in milliseconds. Other aspects of cryo-TEM, such as different cooling techniques, specimen preparation and different [517] methods involved in imaging of vitrified specimens are reviewed elsewhere [435, 436] . The interaction of several AMPs with model membranes or cell membranes has been studied by this technique [437e440] . The Human islet amyloid polypeptide (hIAPP) fibrillar deposits on pancreatic islets of Langerhans cells are thought to be involved in the death of the insulin-producing islet cells in type 2 Diabetes mellitus. Investigation of cryo-TEM analysis of hIAPP with LUVs revealed that hIAPP fibrils distorted LUVs into noncircular shapes [441] . Similarly, Edwards and co-workers [442] used cryo-TEM and other complementary techniques to explore the membrane perturbing effects of melittin and alamethicin on POPC based liposomes of varying composition. They observed that both the LPs induced holes and open bilayer structures on the membrane at higher concentrations.

Unlike TEM (passing a flood of electron beams), SEM moves a spot of the electron beam on the surface of the sample in a raster manner ('to and fro' in a horizontal pattern). The electrons reflected from the surface of the sample are captured by the detector to form the image. The SEM resolution is determined by the electrons spot diameter and not by the wavelength of the electron, as in the case of TEM. The working principles and specimen preparation of SEM have already been reviewed [455e458]. SEM can yield information about surface topography, crystalline structure, chemical composition and nanostructures formed by peptides (Table 11 ). The study of antimicrobial activity of hybrid AMPs (LI) derived from combinations of the typical fragment of human cathelicidin-derived LL37 with indolicidin, revealed that LI exhibited higher antimicrobial activity and cell selectivity than the parental molecules [459] . SEM analysis of membrane morphology of E. coli and Staphylococcus aureus upon LI treatment showed significant membrane damage. The membrane surface of peptide-treated E. coli cells became shrunken, broken and outstretched. Kondorosi and co-workers [460] studied the antibacterial activities of nodule-specific cysteine-rich (NCR) small peptides such as NCR247, NCR335, polymyxin B (PMB) and streptomycin. SEM analysis revealed that complete cell disruption was induced by PMB and NCR335 in Salmonella enterica, while NCR247 treatment resulted in extensive S. enterica cell surface budding. SEM was used to examine the ultrastructural changes induced by b-stranded gramicidin S and the a-helical peptidyl-glycylleucine-carboxy amide (PGLa) in bacteria [461] . SEM images of E. coli cells exposed to these AMPs revealed shortening and swelling of the cells. Also, multiple blisters and bubbles were formed on their surface. The S. aureus cells seemed to burst, showing open holes and deep craters in their cellular envelope.

Detailed information about peptide self-assembly and formation of different nanostructures, such as tubular structures, fibers, micelles, vesicles, and spherical and rod-coil structures can be obtained by SEM. Yang and coworkers [462] designed a short amphiphilic peptide (CG3R6TAT), and studied its ability to form core-shell structured nanoparticles (micelles). Using Field Emission SEM (FE-SEM), they observed nanoparticles of the peptide with sizes smaller than 150 nm. Recently, the development of antibacterial applications using self-assembled peptide gels made of ultrashort peptides gained the attention of scientists [463, 464] . Peptide gels formed from natural amino acids sequences have improved stability against proteolytic enzymes. Through SEM it is possible to visualize the surface morphology of peptide gels. For example, Singh and co-workers [465] reported two a/g hybrid peptides Boc-D-Phe-g 4 -L-Phe-PEA (NH007) and Boc-L-Phe-g 4 -L-Phe-PEA (NH009) which can self-assemble into gels in DMSO solution. SEM images of dried gels of NH007 and NH009 exhibited porous morphologies (an interwoven morphology for NH007 and a flake-like network for NH009). In another study, Banerjee and coworkers [466] synthesized a series of peptides linked to fatty acid chains on their C-terminal amino acid and a free amino group (on the N-terminal). These can self-assemble in aqueous medium prompted by various noncovalent interactions. The FE-SEM images of xerogels of the peptides showed the formation of intertwined nanofibrillar assemblies in all the hydrogel matrices.

Fluorescence microscopy (FM) has provided valuable details about lipid domains in bilayers that were unavailable before [477e479]. The continued development of different FM procedures, such as confocal microscopy, one-photon, and two-photon approaches have offered more information about membrane physiology. Due to the rapid advancement in the bio-photonics field, it is now possible to carry out Fluorescence Correlation Spectroscopy (FCS), three-dimensional particle tracking methods, including polarization, lifetime and emission spectra in a microscopic environment ( . It is also possible to study the lateral structure of membranes and the distribution of peptides on the membrane (provided that the peptide is fluorescently labeled). Using confocal microscopy, Yamazaki and co-workers [494] studied the interaction of carboxyfluorescein (CF)-labeled magainin 2 (CF-magainin 2) with single GUVs containing a water-soluble fluorescent probe, Alexa Fluor 647 hydrazide (AF647). The fluorescence intensity of the rim of the GUV was increased due to the CF-magainin 2 adsorption before AF647started leaking. Andresen and co-workers [495] used FMbased single-vesicle detection methods to study the interaction of mastoparan X, melittin and magainin 2 with POPC/POPG (3:1) LUVs. Confocal imaging of surface-immobilized LUVs revealed that some LUVs were completely emptied of their contents, while some were only partly emptied. In another study, time-lapse fluorescence lifetime imaging microscopy was used to investigate the interaction between fluorescent melittin analogs with single giant unilamellar vesicles [496] . Recently, Yamazaki and co-workers [497] investigated the interaction of the CF-labeled transporter 10 (CF-TP10) with single GUVs. These vesicles were formed with DOPG and DOPC and contained AF647. Through confocal FM, they observed leakage of AF647 at higher concentrations of CF-TP10. Total-Internal-Reflection Fluorescence (TIRF) microscopy is another variation of FM. It can be used to image peptides in live cells or on supported lipid bilayers. When immobilized on surfaces, this technique allows single-molecule level resolution. Using this method, the residence time histogram of the individual peptide on the membrane can be determined. Hence, the binding rate of a peptide onto the membrane can be studied. In TIRF microscopy, the excitation wave impinges on a transparent solid (e.g., cover glass), this process creates an electromagnetic wave, called evanescent wave just over the glass and its interface with the liquid. This evanescent wave has the same frequency as the excited light. Since the evanescent light has limited penetration (~20e200 nm) into the solution, the background signals from molecules in bulk solution are not collected. In all cases, the cells are on the glass and then in the evanescent field. This feature offers TIRF five to-ten-fold better resolution than confocal microscopy. Harris and co-workers [498] used the TIRF microscopic method to characterize membrane affinity of the glucagon-like peptide-1 (GLP-1), a 30-residue membrane-active peptide. TIRF microscopy is generally used in combination with AFM, allowing the recollection of information about the local structure, dynamics and conformational requirements of the peptide-lipid interaction. Combined AFM and polarized TIRF microscopy for investigation on the interaction of the PFWRIRIRR-amide AMP with bacterial membrane-mimetic supported phospholipid bilayers, composed of POPE/tetraoleoyl cardiolipin (TOCL), was carried out by Yip et al., [499, 500] . The TIRF images revealed the TOCL structure perturbation after the introduction of the peptide. A similar method was used by the same group to study the interaction between indolicidin and eukaryotic (DOPC: DSPC: cholesterol) and prokaryotic (DOPE/DOPG) model membranes [501] .

Differential scanning calorimetry (DSC) is an effective and nonperturbing analytical tool to study the thermotropic properties of LP-lipid interactions (Table 13 ). DSC is one of the earliest analytical methods developed for biological samples. In the conventional up scanning mode DSC instruments monitor the temperature difference between a reference cell that is filled with solvent and a sample cell that contains the lipid/peptide of interest in an identical solvent. As the temperature of both cells is increased, thermally induced processes in the sample would initiate the temperature difference relative to the reference cell. Heaters on the sample cell surface supply additional heat to return the temperature difference to its initial value. This additional heat is proportional to the excess heat capacity of the thermally induced process. A single heat absorption peak is often observed in the DSC scan, or 'thermogram'. After correction of instrumental baseline, the peak can be integrated to give a direct calorimetric measurement of the enthalpy for the process (DHcal) and a measure of the melting temperature (Tm).

As discussed earlier, change in the phase transition can reveal details about the peptide-lipid interaction. DSC can provide quantitative information of peptide interaction on membrane structure by comparing the thermotropic data of the lipid blank and the sample with the peptide in a concentration dependent manner [518] . Furthermore, the role of membrane perturbation and surface defects caused by protein-lipid interactions has implicated AMP activity based on phase separation, charge-charge interaction, membrane curvature strain, pore formation, and even detergent style effects [518] .

DSC has been a crucial tool for studying the preferential interaction of AMPs with certain lipid components. Studies with different AMPs have shown a preference binding towards PG as opposed to other negatively charged lipids, such as, phosphatidic acid, phosphatidylserine, and cardiolipin [519] . Phase separation was constantly observed with the different peptides and PE/PG mixtures, with a preferential interaction with PG. It has been evident from DSC studies that lipid specificity is not purely electrostatic and other membrane properties can contribute to the lipid specificity. The DSC study of the interaction of protegrin-1 (PG-1) with phospholipid model membranes suggested that in addition to an overall negative charge, the structural features of the phospholipid headgroups, lipid packing, and thus, membrane fluidity influence the interaction with PG-1 [520, 521] . DSC was also used to show PG lipid segregation upon interaction with AMPs. DSC analysis of AMPs, such as, FRWWHR, RAWVAWR-NH2 and cathelicidin with model membranes showed phase transition behavior [522e524].

Along with scattering techniques and NMR, isothermal titration calorimetry (ITC) has proven to be a reliable tool for providing valuable insights into thermodynamic characterization of AMPsmembrane interactions (Table 13) . A single calorimetric titration can provide a wealth of information such as enthalpy of binding (DH), entropy of binding (DS), association constant (Ka), binding stoichiometry (n), free energy of binding (DG), and potential siteesite interactions. ITC is robust, and can measure binding affinities as low as~100 mM to 1 nM. ITC can measure high affinity interactions, which is not easily achieved with other label-free methods. For instance, radiolabeling techniques and fluorescence spectroscopy can be used to measure high affinity binding, but require the judicious introduction of highly sensitive labels at a specific site. In an ITC experiment, a solution of one compound is titrated against the solution of another compound in an isothermal measurement. The reference cell and the sample cell are set to the desired experimental temperature. The injection device is inserted into the sample cell containing the protein of interest. A series of small aliquots of ligand are injected into the protein solution. In the event ligand-protein binding, heat changes of a few millionths of a degree Celsius are detected and measured. As the first injection is made, the microcalorimeter measures all heat released until the binding reaction has reached equilibrium. The quantity of heat measured is in direct proportion to the amount of binding.

In the event of LP-lipid interactions, ITC can reveal details about secondary processes accompanying peptide-membrane binding, that is, peptide-induced membrane permeabilizations, lipid phase changes, membrane-induced peptide-peptide associations, protonation reactions at the membrane surface and peptide conformational changes. ITC analysis of magainin 2 and PGLa has provided a lot of information about thermodynamics or membrane association [525e530]. A quantitative analysis of the ITC traces reveals enthalpies of 3e4 kcal/mol for PGLa and magainin, respectively, entropies of 40 cal mol À1 K À1 , and apparent membrane association constants in the 106 M À1 range. A recent ICT study of the AMP NK-2 with phospholipid membranes reveled that the overall binding reaction was endothermic [531] . The lipid to NK-2 (L/NK-2) ratio at which saturation of the heat flow occurs was 20:1. In another study, the thermodynamics of the binding process of LAH4-L1, a synthetic amphipathic peptide was elucidated with isothermal titration calorimetry. The ITC data revealed that the binding process was composed of at least three different reactions, that is, a coil-to-helix transition with an exothermic enthalpy of about À11 kcal/mol and two endothermic processes with enthalpies of~4 and~8 kcal/mol, respectively, which partly compensated the exothermic enthalpy of the conformational change [532] .

The exact manner by which AMPs perturbs the cell membrane is a primary requirement to design efficient novel antimicrobial or cell-penetrating peptides. The degree of toxicity of a given AMP depends upon its biochemical properties, specific lipid and/or membrane composition affinity and the microenvironment of peptide-membrane interaction event. In the biophysical community, a major challenge is to determine which analytical techniques can provide information about this biologically important process. In order to address this issue, we reviewed the principal analytical methods that can give detailed information about lipid-peptide interactions (Fig. 6 ). Based on these analytical methods, multidimensional analysis of peptide-lipid interactions must be done in the future. The articulation of the results from various analytical techniques can give a complete perspective on peptide-membrane interactions at the macro and microscopic level.

In conclusion, the understating of the distinct analytical techniques and their involvement in the interaction between LPs and cell membranes at the molecular level, and more specifically at the atomic level, is crucial in the field of drug development. Proper knowledge of these techniques can help design molecules with tailored functionalities. These data are particularly important for antibiotic and therapeutic advancement. Besides analyzing peptide synthesis, new screening on natural fronts and model membrane preparation, understating the biophysical bases of the interaction between natural bioactive molecules and the cell membrane is of utmost importance.

S.M and C.M.G conceived of the presented idea. R.C and B.B participated with manuscript content and organization. All authors provided critical feedback and helped shape the research, analysis and manuscript.

Biosurfactants: multifunctional biomolecules of the 21st century

Applications of a lipopeptide biosurfactant, surfactin, produced by microorganisms

Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases, Microbiol

LPs as antiinfectives: a practical perspective

Structure of the lipopeptide antibiotic tsushimycin

On glumamycin, a new antibiotic. vi. an approach to the amino acid sequence

On glumamycin, a new antibiotic

The steric configuration of a, b -diaminobutyric acid

Activity against experimental infections in mice

Structure determination of fatty acids from the antibiotic aspartocin

Natural products to drugs: daptomycin and related lipopeptide antibiotics

Laspartomycin, an acidic lipopeptide antibiotic with a unique peptide core

Molecular biology of antibiotic production in Bacillus

Suppression of inflammatory responses by surfactin, a selective inhibitor of platelet cytosolic phospholipase A2

Leakage and lysis of lipid membranes induced by the lipopeptide surfactin

Chemical composition of crystallomycin

NMR evidence for a peptide antibiotic with folded structure in water

Solution structure of polymyxins B and E and effect of binding to lipopolysaccharide: an NMR and molecular modeling study

The structure of octapeptin D (studies on antibiotics from the genus Bacillus. XXVIII)

Mechanism of membrane activity of the antibiotic trichogin GA IV: a twostate transition controlled by peptide concentration

Effect of peptide lipidation on membrane perturbing activity: a comparative study on two trichogin analogues

Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics

Tryptophan-and arginine-rich antimicrobial peptides: structures and mechanisms of action

The medicinal chemistry of short lactoferricin-based antibacterial peptides

Combinatorial biosynthesis of novel antibiotics related to daptomycin

A bactericidal effect for human lactoferrin

Bestowing antifungal and antibacterial activities by lipophilic acid conjugation to d,l-amino acid-containing antimicrobial peptides: a plausible mode of action

A new group of antifungal and antibacterial LPs derived from non-membrane active peptides conjugated to palmitic acid

Acyl-substituted dermaseptin S4 derivatives with improved bactericidal properties, including on oral microflora

Ultrashort antibacterial and antifungal LPs

Synthetic LPs: a novel class of anti-infectives

Constraining cyclic peptides to mimic protein structure motifs

Production of cyclic LPs by fluorescent pseudomonads

Relevance of chlorine-substituent for the antifungal activity of syringomycin and syringotoxin, metabolites of the phytopathogenic bacterium Pseudomonas syringae pv. syringae

Identification of a biosynthetic gene cluster and the six associated LPs involved in swarming motility of Pseudomonas syringae pv. tomato DC3000

Bioactive LPs of ice-nucleating snow bacterium Pseudomonas syringae strain 31R1

Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria

Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli

Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin

Mode of action of the new antibiotic for Grampositive pathogens daptomycin: comparison with cationic antimicrobial peptides and LPs

Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus

Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

Promotion of peptide antimicrobial activity by fatty acid conjugation

Acylation of SC4 dodecapeptide increases bactericidal potency against Gram-positive bacteria, including drug-resistant strains

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Biosurfactants: properties, commercial production and application

Isolation and characterization of two new lipopeptide biosurfactants produced by Pseudomonas fluorescens BD5 isolated from water from the Arctic Archipelago of Svalbard

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25

Improved performance of cross-flow ultrafiltration for the recovery and purification of Ca 2þ conditioned LPs in diafiltration mode of operation

Isolation and characterization of a lipopeptide bioemulsifier produced by Pseudomonas nitroreducens TSB.MJ10 isolated from a mangrove ecosystem

An inexpensive strategy for facilitated recovery of metals and fermentation products by foam fractionation process

Integrated process for production of surfactin: Part 2. Equilibrium and kinetic study of surfactin adsorption onto activated carbon

Evaluation of antagonistic activities of Bacillus subtilis and Bacillus licheniformis against wood-staining fungi: in vitro and in vivo experiments

Screening concepts, characterization and structural analysis of microbial-derived bioactive LPs: a review

Engineering lipid bilayer membranes for protein studies

Getting across the cell membrane: an overview for small molecules, peptides, and proteins

Computational modeling of realistic cell membranes

Tethered and polymer supported bilayer lipid membranes: structure and function

A trough for improved SFG spectroscopy of lipid monolayers

The application of biophysical techniques to study antimicrobial peptides

Structure and phase transitions in Langmuir monolayers

Biophysical characterization of the insertion of two potent antimicrobial peptides-Pin2 and its variant Pin2[GVG] in biological model membranes

Evaluation of lipopeptide (daptomycin) aggregation using fluorescence, light scattering, and nuclear magnetic resonance spectroscopy

Structure, membrane orientation, mechanism, and function of pexiganan d a highly potent antimicrobial peptide designed from magainin

Action of antimicrobial peptides: two-state model

Mode of action of membrane active antimicrobial peptides

Peptideemembrane interactions and mechanisms of membrane destruction by amphipathic a-helical antimicrobial peptides

A glimpse of structural biology through X-ray crystallography

Phase determination for membrane diffraction by anomalous dispersion

Chain packing in the inverted hexagonal phase of phospholipids: a study by X-ray anomalous diffraction on bromine-labeled chains

Peptides in lipid bilayers: determination of location by absolute-scale X-ray refinement

Novel methods for studying lipids and lipases and their mutual interaction at interfaces: Part II. Surface sensitive synchrotron X-ray scattering

Elements of Modern X-Ray Physics

Action of antimicrobial peptides: two-state model

In situ characterization of lipid A interaction with antimicrobial peptides using surface X-ray scattering

A comparative study on the interactions of SMAP-29 with lipid monolayers

Sigmoidal concentration dependence of antimicrobial peptide activities: a case study on alamethicin

Membrane pores induced by magainin

Structure, location, and lipid perturbations of melittin at the membrane interface

Chain packing in the inverted hexagonal phase of phospholipids: a study by X-ray anomalous diffraction on bromine-labeled chains

Small-angle X-ray scattering on biological macromolecules and nanocomposites in solution

Interaction of the lipopeptide biosurfactant lichenysin with phosphatidylcholine model membranes

Interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity

Structural diversity and mode of action on lipid membranes of three lactoferrin candidacidal peptides

Structure of the alamethicin pore reconstructed by X-ray diffraction analysis

Cation-selective pathway of OmpF porin revealed by anomalous X-ray diffraction

Cation-selective pathway of OmpF porin revealed by anomalous X-ray diffraction

Magainin 2 in phospholipid bilayers: peptide orientation and lipid chain ordering studied by X-ray diffraction

Lipid headgroup discrimination by antimicrobial peptide LL-37: insight into mechanism of action

Amyloid-b fibrillogenesis seeded by interface-induced peptide misfolding and self-assembly

Antimicrobial peptide mimics as potential anticancer agents:interactions of acyl-lysine oligomer C12K-7Alpha8 with ganglioside/DPPC mixtures

In situ characterization of lipid A interaction with antimicrobial peptides using surface X-ray scattering

K€ as, Interaction of the MARCKS peptide with PIP2 in phospholipid monolayers

A comparative study on the interactions of SMAP-29 with lipid monolayers

Mechanism of membrane perturbation by the HIV-1 gp41 membrane-proximal external region and its modulation by cholesterol

Protegrin interaction with lipid monolayers: grazing incidence X-ray diffraction and X-ray reflectivity study

Structure of magainin and alamethicin in model membranes studied by X-ray reflectivity

Alamethicin in lipid bilayers: combined use of X-ray scattering and MD simulations

Amyloid b-peptide insertion in liposomes containing GM1-cholesterol domains

Comparative structural studies of vpu peptides in phospholipid monolayers by X-ray scattering

Synergism of antimicrobial frog peptides couples to membrane intrinsic curvature strain

Membrane thickening by the antimicrobial peptide PGLa

Andr€ a, Structural rearrangement of model membranes by the peptide antibiotic NK-2

Aggregation structure of Alzheimer amyloid-b(1e40) peptide with sodium dodecyl sulfate as revealed by small-angle X-ray and neutron scattering

Helical 1:1 a/Sulfono-g-AA heterogeneous peptides with antibacterial activity

HIV-1 tat membrane interactions probed using X-ray and neutron scattering, CD spectroscopy and MD simulations

CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol

ATR-FTIR studies in pore forming and membrane induced fusion peptides

Attenuated total reflection IR spectroscopy as a tool to investigate the orientation and tertiary structure changes in fusion proteins

ATR-FTIR: a "rejuvenated" tool to investigate amyloid proteins

The chain order of binary unsaturated lipid bilayers modulated by aromatic-residue-containing peptides: an ATR-FTIR spectroscopy study

Attenuated total reflection infrared spectroscopy of proteins and lipids in biological membranes

Amide modes and protein conformation

Relevance of protein thin films prepared for attenuated total reflection fourier transform infrared spectroscopy: significance of the pH

HydrogenÀDeuterium exchange of streptavidin and its complex with biotin studied by 2D-attenuated total reflection fourier transform infrared spectroscopy

Membrane interactions of a self-assembling model peptide that mimics the self-association, structure and toxicity of Ab(1e40)

Formation and structure of surface films: captive bubble surfactometry

In situ measurement of the infrared spectra of insoluble monolayers at the airewater interface

Quantitative external reflection infrared spectroscopic analysis of insoluble monolayers spread at the airewater interface

In situ measurement of the FT-IR spectra of phospholipid monolayers at the air/water interface

External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental design, spectra-structure correlations, and effects of hydrogendeuterium exchange

Investigations at the air/water interface using polarization modulation IR spectroscopy

Infrared and Raman spectroscopies of monolayers at the airewater interface

Protein-lipid interactions at the air/water interface

Specific interaction restrains structural transitions of an amphiphilic peptide in pulmonary surfactant model systems: an in situ PM-IRRAS investigation

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Amyloid-b fibrillogenesis seeded by interface-induced peptide misfolding and self-assembly

Structure and interaction with lipid membrane models of semliki forest virus fusion peptide

Binding of cationic model peptides (KX)4K to anionic lipid bilayers: lipid headgroup size influences secondary structure of bound peptides

The transmembrane domain of the SNARE protein VAMP2 is highly sensitive to its lipid environment

Membrane docking of the C2 domain from protein kinase ca as seen by polarized ATR-IR. The role of PIP2

Galleria mellonella native and analogue peptides Gm1 and DGm1. I) biophysical characterization of the interaction mechanisms with bacterial model membranes

The human cathelicidin LL-37 d a pore-forming antibacterial peptide and hostcell modulator

Interaction of a peptide derived from C-terminus of human TRPA1 channel with model membranes mimicking the inner leaflet of the plasma membrane

Dual functions of the human antimicrobial peptide LL-37dtarget membrane perturbation and host cell cargo delivery

Surface pressure induced structural transitions of an amphiphilic peptide in pulmonary surfactant systems by an in situ PM-IRRAS study

Location of structural transitions in an isotopically labeled lung surfactant SP-B peptide by IRRAS

Interfacial properties and structure stability of the gp41 tryptophan-rich peptide from HIV-1

The importance of cyclic structure for labaditin on its antimicrobial activity against Staphylococcus aureus

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the airewater interface

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the airewater interface

Surface activity and structures of two fragments of the human antimicrobial LL-37

Ideally amphipathic b-sheeted peptides at interfaces: structure, orientation, affinities for lipids and hemolytic activity of (KL)mK peptides

Influence of the 524-VAAEIL-529 sequence of annexins A6 in their interfacial behavior and interaction with lipid monolayers

Influence of the 524-VAAEIL-529 sequence of annexins A6 in their interfacial behavior and interaction with lipid monolayers

Fluorescence spectroscopy methodologies on the study of proteins and peptides. On the 150th anniversary of protein fluorescence

Interaction of the major epitope region of HIV protein gp41 with membrane model systems. A fluorescence spectroscopy study

Self-association of the polyene antibiotic nystatin in dipalmitoylphosphatidylcholine vesicles: a time-resolved fluorescence study

Evaluation of the membrane lipid selectivity of the pea defensin Psd1

Vesicle leakage reflects the target selectivity of antimicrobial LPs from Bacillus subtilis

Influence of the bilayer composition on the binding and membrane disrupting effect of polybia-MP1, an antimicrobial mastoparan peptide with leukemic Tlymphocyte cell selectivity

Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L

Fluorescence quenching at interfaces and the permeation of acrylamide and iodide across phospholipid bilayers

Outer membrane protein A of Escherichia coli inserts and folds into lipid bilayers by a concerted mechanism

Outer membrane protein A of Escherichia coli inserts and folds into lipid bilayers by a concerted mechanism

Multiple binding sites for fatty acids on the potassium channel KcsA

Effects of lipid structure on the state of aggregation of potassium channel KcsA

Characterizing the fatty acid binding site in the cavity of potassium channel KcsA

Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening

A fluorescence method to define transmembrane ahelices in membrane proteins: studies with bacterial diacylglycerol kinase

Fluorescence quenching methods to study lipid-protein interactions

Characterization of daptomycin oligomerization with perylene excimer fluorescence: stoichiometric binding of phosphatidylglycerol triggers oligomer formation

Oligomerization of daptomycin on membranes

Pyrene: a probe to study protein conformation and conformational changes

Ionic-strengthand pH-dependent conformational states of human plasma fibronectin

Human plasma fibronectin structure probed by steady-state fluorescence polarization: evidence for a rigid oblate structure

Solvent effect on the vibrational structures of the fluorescence and absorption spectra of pyrene

Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems

Pyrene fluorescence analysis offers new insights into the conformation of the lipoprotein-binding domain of human apolipoprotein E

Membrane binding and oligomerization of the lipopeptide A54145 studied by pyrene fluorescence, Biophys

Liposome-cell interaction: transfer and intracellular release of a trapped fluorescent marker

Mechanism of action and limited cross-resistance of new lipopeptide MX-2401

Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism

A novel antimicrobial peptide, scolopendin, from Scolopendra subspinipes mutilans and its microbicidal mechanism

Determination of ion permeability by fluorescence quenching

Structural features of model glycopeptides in solution and in membrane phase: a spectroscopic and molecular mechanics investigation

Tryptophancontaining lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram-negative bacteria

Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin

Fluorescence indicates a calcium-dependent interaction between the lipopeptide antibiotic LY146032 and phospholipid membranes

Lipid-specific binding of the calcium-dependent antibiotic daptomycin leads to changes in lipid polymorphism of model membranes

Oligomerization of daptomycin on membranes

Estimation of the subunit stoichiometry of the membrane-associated daptomycin oligomer by FRET

On the quest for the elusive mechanism of action of daptomycin: binding, fusion, and oligomerization

Fluorescence correlation spectroscopy of molecular motions and kinetics

Protein-Protein Interact

Recent applications of fluorescence correlation spectroscopy in live systems

Fluorescence correlation spectroscopy: past, present, future, Biophys

Fluorescence correlation spectroscopy: linking molecular dynamics to biological function in vitro and in situ

Binding of the Alzheimer amyloid b-peptide to neuronal cell membranes by fluorescence correlation spectroscopy

Measurement of the attachment and assembly of small amyloid-b oligomers on live cell membranes at physiological concentrations using single-molecule tools

Interaction of MreB-derived antimicrobial peptides with membranes

Preferential binding of apolipoprotein E derived peptides with oxidized phospholipid

Secondary structure, phospholipid membrane interactions, and fusion activity of two glutamate-rich analogs of influenza hemagglutinin fusion peptide

Interaction of LDS-751 with the drug-binding site of P-glycoprotein: a trp fluorescence steady-state and lifetime study

Effect of N-terminal acetylation on lytic activity and lipid-packing perturbation induced in model membranes by a mastoparan-like peptide

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide pep-1

Investigating the cationic side chains of the antimicrobial peptide tritrpticin: hydrogen bonding properties govern its membrane-disruptive activities

Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against gramnegative and gram-positive bacteria

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptideemembrane interactions

Elucidating the mechanism of peptide interaction with membranes using the intrinsic fluorescence of tryptophan: perpendicular penetration of cecropin B-like peptides into Pseudomonas aeruginosa

Dynamic turn conformation of a short tryptophan-rich cationic antimicrobial peptide and its interaction with phospholipid membranes

Interaction of synthetic peptides corresponding to hepatitis G virus (HGV/GBV-C) E2 structural protein with phospholipid vesicles

Biophysical evaluation of cardiolipin content as a regulator of the membrane lytic effect of antimicrobial peptides

Nucleation and growth of pores in 1,2-dimyristoyl-Sn-Glycero-3-Phosphocholine (DMPC)/Cholesterol bilayer by antimicrobial peptides melittin, its mutants and cecropin P1

Membrane interactions and biological activity of antimicrobial peptides from Australian scorpion

Proline-15 creates an amphipathic wedge in maculatin 1.1 peptides that drives lipid membrane disruption

Studies of membranotropic and fusogenic activity of two putative HCV fusion peptides

Highly disordered amyloid-b monomer probed by single-molecule FRET and MD simulation

Comparison of lipid-dependent bilayer insertion of pHLIP and its P20G variant

Membrane perturbation by the antimicrobial peptide PMAP-23: a fluorescence and molecular dynamics study

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy studies of peptide and protein binding to phospholipid vesicles

Investigation of a novel artificial antimicrobial peptide by fluorescence correlation spectroscopy: an amphipathic cationic pattern is sufficient for selective binding to bacterial type membranes and antimicrobial activity

Peptide conformation and protein folding

Responsive gels formed by the spontaneous selfassembly of peptides into polymericb-sheettapes

Folding propensity and biological activity of peptides: the effect of a single stereochemical isomerization on the conformational properties of bombinins in aqueous solution

Design, structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

Mechanism of lipid bilayer disruption by the human antimicrobial peptide

Molecular state of the membrane-active antibiotic daptomycin

Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

Dynamical structure of the short multifunctional peptide BP100 in membranes

Lipid-alamethicin interactions influence alamethicin orientation

Mechanism of alamethicin insertion into lipid bilayers

Effect of changing the size of lipid headgroup on peptide insertion into membranes

Sigmoidal concentration dependence of antimicrobial peptide activities: a case study on alamethicin

Barrel-stave model or toroidal model? A case study on melittin pores

Energetics of pore formation induced by membrane active peptides

Cooperative membrane insertion of magainin correlated with its cytolytic activity

Sigmoidal concentration dependence of antimicrobial peptide activities: a case study on alamethicin

Effect of changing the size of lipid headgroup on peptide insertion into membranes

Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat

Effect of membrane composition on antimicrobial peptides aurein 2.2 and 2.3 from Australian southern bell frogs

Family of pH (low) insertion peptides for tumor targeting

Insertion into lipid bilayer of truncated pHLIP® peptide

Oriented circular dichroism: a method to characterize membrane-active peptides in oriented lipid bilayers

Fast and potent bactericidal membrane lytic activity of PaDBS1R1, a novel cationic antimicrobial peptide

Antimicrobial activity and self-assembly behavior of antimicrobial peptide chensinin-1b with lipophilic alkyl tails

Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. Pneumon iae and P. Aeruginosa

Lipopolysaccharide induces amyloid formation of antimicrobial peptide HAL-2

Antimicrobial effects of novel peptides cOT2 and sOT2 derived from Crocodylus siamensis and pelodiscus sinensis ovotransferrins

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms

Solvent-dependent structure of two tryptophan-rich antimicrobial peptides and their analogs studied by FTIR and CD spectroscopy

Structural and functional evaluation of the palindromic alanine-rich antimicrobial peptide Pa-MAP2

Structural and biophysical characterization of an antimicrobial peptide chimera comprised of lactoferricin and lactoferrampin

Antibacterial properties of human beta defensin-3 derivative: CHRG01

Identification, recombinant expression, and characterization of LGH2, a novel antimicrobial peptide of Lactobacillus casei HZ1

Concentration-dependent, membrane-selective activity of human LL37 peptides modified with collagen binding domain sequences

Arginine-containing surfactant-like peptides: interaction with lipid membranes and antimicrobial activity

High level and purification of the clinically active antimicrobial peptide P-113 in Escherichia coli

Antimicrobial activity and self-assembly behavior of antimicrobial peptide chensinin-1b with lipophilic alkyl tails

Importance of residue 13 and the C-terminus for the structure and activity of the antimicrobial peptide aurein 2.2

The interaction of melittin with dimyristoyl phosphatidylcholine-dimyristoyl phosphatidylserine lipid bilayer membranes

Interactions of lipopolysaccharide and polymyxin studied by NMR spectroscopy

Mapping residue-specific contacts of polymyxin B with lipopolysaccharide by saturation transfer difference NMR: insights into outer-membrane disruption and endotoxin neutralization

Mapping the targeted membrane pore formation mechanism by solution NMR: the Nisin Z and lipid II interaction in SDS micelles

High-resolution NMR studies of antibiotics in cellular membranes

Choosing membrane mimetics for NMR structural studies of transmembrane proteins

Testing membrane interactions of CPPs

Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions

NMR studies of three-dimensional structure and positioning of CPPs in membrane model systems

Artificial membrane models for the study of macromolecular delivery

Spontaneous formation of stable unilamellar vesicles

Generation of multilamellar and unilamellar phospholipid vesicles

Vesicles of variable sizes produced by a rapid extrusion procedure

Bicelles as model membranes for solid-and solutionstate NMR studies of membrane peptides and proteins

Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins

Solution NMR of membrane proteins: practice and challenges

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Location of M13 coat protein in sodium dodecyl micelles as determined by NMR

Three-dimensional structure and position of porcine motilin in sodium dodecyl sulfate micelles determined by 1H NMR

Biophysical approaches to membrane protein structure determination

Isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution NMR studies of polypeptides

Bicelles: a model membrane system for all seasons?

Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies

Methods for studying transmembrane peptides in bicelles: consequences of hydrophobic mismatch and peptide sequence

The membrane-induced structure of melittin is correlated with the fluidity of the lipids

Static and dynamic properties of phospholipid bilayer nanodiscs

Chapter Eleven -Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs

Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins

Lipideprotein nanodiscs offer new perspectives for structural and functional studies of water-soluble membrane-active peptides

Nanodiscs as a New Tool to Examine LipideProtein Interactions BT -Lipid-Protein Interactions: Methods and Protocols

Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins

LipidÀProtein nanodiscs as reference medium in detergent screening for high-resolution NMR studies of integral membrane proteins

Two-dimensional spectroscopy. Application to nuclear magnetic resonance

Coherence transfer by isotropic mixing: application to proton correlation spectroscopy

Vesicle-bound conformation of melittin: transferred nuclear Overhauser enhancement analysis in the presence of perdeuterated phosphatidylcholine vesicles

Dimer structure of magainin 2 bound to phospholipid vesicles

Interaction of the dengue virus fusion peptide with membranes assessed by NMR: the essential role of the envelope protein Trp101 for membrane fusion

Helical structure and orientation of melittin in dispersed phospholipid membranes from amide exchange analysis in situ

A kinked antimicrobial peptide from Bombina maxima. I. Three-dimensional structure determined by NMR in membrane-mimicking environments

The Use of Residual Dipolar Coupling in Studying Proteins by NMR BT -NMR of Proteins and Small Biomolecules

The use of residual dipolar coupling in studying proteins by NMR

Determination of helical membrane protein topology using residual dipolar couplings and exhaustive search algorithm: application to phospholamban

Dipolar waves as NMR maps of protein structure

Dipolar waves map the structure and topology of helices in membrane proteins

Orientation and conformational preference of leucine-enkephalin at the surface of a hydrated dimyristoylphosphatidylcholine bilayer: NMR and MD simulation

Location and orientation relative to the micelle surface for glucagon in mixed micelles with dodecylphosphocholine EPR and NMR studies

Oxygen as a paramagnetic probe of membrane protein structure by cysteine mutagenesis and 19 F NMR spectroscopy

The measurement of immersion depth and topology of membrane proteins by solution state NMR

Determination of membrane immersion depth with O(2): a high-pressure (19)F NMR study

Using O(2) to probe membrane immersion depth by (19)F NMR

Membrane proteinelipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents

S€ oderh€ all, Interaction of the antimicrobial peptide cyclo (RRWWRF) with membranes by molecular dynamics simulations

NMR studies of lysozyme surface accessibility by using different paramagnetic relaxation probes

Mapping the orientation of helices in micelle-bound peptides by paramagnetic relaxation waves

Determining the orientation and localization of membrane-bound peptides

Positioning of micelle-bound peptides by paramagnetic relaxation enhancements

Structures of b-hairpin antimicrobial protegrin peptides in lipopolysaccharide membranes: mechanism of gram selectivity obtained from solid-state nuclear magnetic resonance

Structure and mechanism of beta-hairpin antimicrobial peptides in lipid bilayers from solid-state NMR spectroscopy

Solid-state nuclear magnetic resonance characterization of gramicidin channel structure

Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy

Membrane structure and interactions of human catestatin by multidimensional solution and solid-state NMR spectroscopy

Structure and alignment of the membraneassociated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy

Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy

Beyond NMR spectra of antimicrobial peptides: dynamical images at atomic resolution and functional insights

Analysis of the amide 15 N chemical shift tensor of the Ca tetrasubstituted constituent of membraneactive peptaibols, the a-aminoisobutyric acid residue, compared to those of di-and tri-substituted proteinogenic amino acid residues

The alignment, structure and dynamics of membrane-associated polypeptides by solid-state NMR spectroscopy

Solution and solid-state NMR structural studies of antimicrobial peptides LPcin-I and LPcin-II

Solid-state NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from magainin 2 and melittin, Biophys

A coiled-coil peptide shaping lipid bilayers upon fusion

NMR and ESR study of amphotericin B interactions with various binary phosphatidylcholine/phosphatidylglycerol membranes

Membrane defects enhance the interaction of antimicrobial peptides

Mechanisms of peptide-induced pore formation in lipid bilayers investigated by oriented 31P solid-state NMR spectroscopy

Membrane interactions in small fast-tumbling bicelles as studied by 31P NMR

Phospholipid head groups as sensors of electric charge in membranes

Solid-state NMR investigations of peptide-lipid interactions of the transmembrane domain of a plant-derived protein, Hcf106

Determining the mode of action involved in the antimicrobial activity of synthetic peptides: a solid-state NMR and FTIR study

Structure and dynamics of the lipid modifications of a transmembrane a-helical peptide determined by 2H solid-state NMR spectroscopy

Action of the multifunctional peptide BP100 on native biomembranes examined by solid-state NMR

Bechinger, pH-dependent membrane interactions of the histidine-rich cell-penetrating peptide LAH4-L1

19F NMR screening of unrelated antimicrobial peptides shows that membrane interactions are largely governed by lipids

Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat

Chapter 11 -applications of 19F magnetic resonance spectroscopy and imaging for the study of nanostructures used in antimicrobial therapy A2 -ficai

19F NMR studies of solvent exposure and peptide binding to an SH3 domain

4-Fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides

Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR

Alamethicin supramolecular organization in lipid membranes from 19 F solid-state NMR

A solid-state NMR index of helical membrane protein structure and topology

Imaging membrane protein helical wheels

Tilt angle of a trans-membrane helix is determined by hydrophobic mismatch

Structural and dynamic insights of the interaction between tritrpticin and micelles: an NMR study

Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2H solid-state NMR

Simulations of membranedisrupting peptides II: AMP piscidin 1 favors surface defects over pores

Role of b-naphthylalanine end-tags in the enhancement of antiendotoxin activities: solution structure of the antimicrobial peptide S1-nalnal in complex with lipopolysaccharide

A 2H solid-state NMR study of lipid clustering by cationic antimicrobial and cell-penetrating peptides in model bacterial membranes

Conformational and membrane interaction studies of the antimicrobial peptide alyteserin-1c and its analogue [E4K]alyteserin-1c

Structureefunction relationships in histidine-rich antimicrobial peptides from atlantic cod

Alanine scan and 2H NMR analysis of the membrane-active peptide BP100 point to a distinct carpet mechanism of action

Structural biology of membrane-acting peptides: conformational plasticity of anticoccidial peptide PW2 probed by solution NMR

The structure and behavior of the NA-CATH antimicrobial peptide with liposomes

Structure and dynamics of the two amphipathic arginine-rich peptides RW9 and RL9 in a lipid environment investigated by solid-state NMR and MD simulations

Proline facilitates membrane insertion of the antimicrobial peptide maculatin 1.1 via surface indentation and subsequent lipid disordering

Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3

Structure of chemokine-derived antimicrobial peptide interleukin-8a and interaction with detergent micelles and oriented lipid bilayers

New limits of sensitivity of site-directed spin labeling electron paramagnetic resonance for membrane proteins

Stoichiometry of lipid-protein interaction and integral membrane protein structure

Stoichiometry of lipid interactions with transmembrane proteinsddeduced from the 3D structures

Spin labeling EPR

Determination of Protein Folds and Conformational Dynamics Using Spin-Labeling EPR Spectroscopy BT -Distance Measurements in Biological Systems by EPR

Three-dimensional architecture and gating mechanism of a Kþ channel studied by EPR spectroscopy

Threedimensional architecture of membrane-embedded MscS in the closed conformation

Location and dynamics of basic peptides at the membrane interface: electron paramagnetic resonance spectroscopy of tetramethyl-piperidine-N-Oxyl-4-Amino-4-Carboxylic acid-labeled peptides

Orientation and lipid-peptide interactions of gramicidin A in lipid membranes: polarized attenuated total reflection infrared spectroscopy and spin-label electron spin resonance

Investigation of model membrane disruption mechanism by melittin using pulse electron paramagnetic resonance spectroscopy and cryogenic transmission electron microscopy

Antimicrobial peptides at work: interaction of myxinidin and its mutant WMR with lipid bilayers mimicking the P. aeruginosa and E. coli membranes

Determining the topology of integral membrane peptides using EPR spectroscopy

Intramembrane water associated with TOAC spin-labeled alamethicin: electron spin-echo envelope modulation by D2O

SARS-CoV fusion peptides induce membrane surface ordering and curvature

Interactions of angiotensin II non-peptide AT1 antagonist losartan with phospholipid membranes studied by combined use of differential scanning calorimetry and electron spin resonance spectroscopy

Membrane disruption mechanism of a prion peptide (106e126) investigated by atomic force microscopy, Raman and electron paramagnetic resonance spectroscopy

Towards nanomicrobiology using atomic force microscopy

Biomolecular imaging with the atomic force microscope

Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces

Phase imaging by atomic force microscopy: analysis of living homoiothermic vertebrate cells

Distribution of ganglioside GM1 between two-component, two-phase phosphatidylcholine monolayers

Blistering of Langmuir-blodgett bilayers containing anionic phospholipids as observed by atomic force microscopy

Nanometer-scale surface properties of mixed phospholipid monolayers and bilayers

Preparation of solid-supported lipid bilayers by spincoating

Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration

AFM characterization of solid-supported lipid multilayers prepared by spin-coating

Phase topology and growth of single domains in lipid bilayers

Structural diversity of sphingomyelin microdomains

Phase behaviour of binary mixtures involving tristearin, stearyl stearate and stearic acid: thermodynamic study and BAM observation at the airewater interface and AFM analysis of LB films

AFM studies on LB films of poly(nhexyl isocyanate), poly(vinyl acetate), and their binary mixtures

Mechanical properties of milk sphingomyelin bilayer membranes in the gel phase: effects of naturally complex heterogeneity, saturation and acyl chain length investigated on liposomes using AFM

A Langmuir and AFM study on interfacial behavior of binary monolayer of hexadecanol/DPPE at the air-water interface

Temperature-controlled structure and kinetics of ripple phases in one-and twocomponent supported lipid bilayers

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions

An AFM study of the effects of silanization temperature, hydration, and annealing on the nucleation and aggregation of condensed OTS domains on mica

Thaulin-1: the first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul

The human cathelicidin LL-37 d a pore-forming antibacterial peptide and hostcell modulator

Biophysical investigations on the interaction between antimicrobial peptides and bacteria killed by Cs-137 irradiation

PSD1 antimicrobial activity against Candida albicans planktonic cells and biofilms

Peptides derived from a-lactalbumin membrane binding helices oligomerize in presence of lipids and disrupt bilayers

Phospholipid dependent mechanism of smp24, an ahelical antimicrobial peptide from scorpion venom

Atomic force microscopy of bacteria reveals the mechanobiology of pore forming peptide action

Mechanisms of antimicrobial peptide action: studies of indolicidin assembly at model membrane interfaces by in situ atomic force microscopy

Membrane selectivity and biophysical studies of the antimicrobial peptide GL13K

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C(14)-peptides

Domain formation in phosphatidylcholine bilayers containing transmembrane peptides: specific effects of flanking residues

Lipidinteracting properties of the N-terminal domain of human apolipoprotein C-III

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Fatty acids can substitute the HIV fusion peptide in lipid merging and fusion: an analogy between viral and palmitoylated eukaryotic fusion proteins

Atomic force microscopy to study molecular mechanisms of amyloid fibril formation and toxicity in Alzheimer's disease

Effects of lipid composition and phase on the membrane interaction of the prion peptide 106e126 amide

PrP106e126 amide causes the semi-penetrated poration in the supported lipid bilayers

Atomic force microscopy characterization of protein fibrils formed by the amyloidogenic region of the bacterial protein MinE on mica and a supported lipid bilayer

Atomic force microscopy under controlled conditions reveals structure of C-terminal region of a-synuclein in amyloid fibrils

Changes in lipid membranes may trigger amyloid toxicity in Alzheimer's disease

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: a combined atomic force microscopy and biochemical study

Effects of the peptide magainin H2 on supported lipid bilayers studied by different biophysical techniques

Phospholipid dependent mechanism of smp24, an ahelical antimicrobial peptide from scorpion venom

Atomic force microscopy of bacteria reveals the mechanobiology of pore forming peptide action

The interfacial properties of the peptide polybia-MP1 and its interaction with DPPC are modulated by lateral electrostatic attractions

Effect of E1(64e81) hepatitis G peptide on the in vitro interaction of HIV-1 fusion peptide with membrane models

Membrane selectivity and biophysical studies of the antimicrobial peptide GL13K

pore formation by a baxderived peptide: effect on the line tension of the membrane probed by AFM

Effects of HIV-1 gp41-derived virucidal peptides on virus-like lipid membranes

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono-and bilayers

Atomic force microscopy study of the antimicrobial action of sushi peptides on gram negative bacteria

Elucidation of mechanisms of interaction of a multifunctional peptide Pa-MAP with lipid membranes

Effect of linker and spacer on the design of a fibronectin-mimetic peptide evaluated via cell studies and AFM adhesion forces

Conventional transmission electron microscopy

The cathelicidin-like peptide derived from panda genome is a potential antimicrobial peptide

Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae

Evaluation of morphological changes of Staphylococcus aureus and Escherichia coli induced with the antimicrobial peptide AN5-1

Imaging the antistaphylococcal activity of CATH-2: mechanism of attack and regulation of inflammatory response

Morphological and functional adaptations of Fusobacterium nucleatum exposed to human neutrophil Peptide-1

Antibacterial effects of Lactobacillus and bacteriocin PLNC8 ab on the periodontal pathogen Porphyromonas gingivalis

Tyrocidine A analogues bearing the planar d-phe-2-abz turn motif: how conformation impacts bioactivity

Negative staining and cryo-negative staining of macromolecules and viruses for TEM

Cryo-TEM of soft molecular assemblies

An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology

Melittin-lipid bilayer interactions and the role of cholesterol

Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers

Bilayer disruption and liposome restructuring by a homologous series of small Arg-rich synthetic peptides

Surfactin-triggered small vesicle formation of negatively charged membranes: a novel membrane-lysis mechanism

Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane

Effect of a-helical peptides on liposome structure: a comparative study of melittin and alamethicin

Structural insight into the mechanism of action of antimicrobial peptide BMAP-28(1e18) and its analogue mutB-MAP18

Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer

Interactions of the antimicrobial peptide Nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity

Antibacterial activity and its mechanisms of a recombinant funme peptide against cronobacter sakazakii in powdered infant formula

The antimicrobial peptide cathelicidin-BF could Be a potential therapeutic for Salmonella typhimurium infection

Structure and membrane interactions of chionodracine, a piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus

Molecular characterization of a novel antimicrobial peptide from Mytilus coruscus

Effective antimicrobial activity of a peptide mutant cbf-14-2 against penicillin-resistant bacteria based on its unnatural amino acids

Characterization of an abaecin-like antimicrobial peptide identified from a pteromaluspuparum cDNA clone

Lipid nanodisc formation using pxt-5 peptide isolated from Amphibian (Xenopus tropicalis) skin, and its altered form, modify-pxt-5

Antimicrobial activity and safety evaluation of peptides isolated from the hemoglobin of chickens

Imaging the antimicrobial mechanism(s) of cathelicidin-2

Sample preparation d the key to SEM studies of failed concrete

Sample preparation for scanning electron microscopy of plant surfacesdhorses for courses

Recent progress in scanning electron microscopy for the characterization of fine structural details of nano materials

Environmental Scanning Electron Microscopy in Cell Biology BT -Cell Imaging Techniques: Methods and Protocols

Characterization of Bactericidal Efficiency, Cell Selectivity, and Mechanism of Short Interspecific Hybrid Peptides

Comparative analysis of the bacterial membrane disruption effect of two natural plant antimicrobial peptides

Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy

Self-assembled cationic peptide nanoparticles as an efficient antimicrobial agent

PLGA nanofibers blended with designer self-assembling peptides for peripheral neural regeneration

Self-assembly of argephe nanostructures via the solidevapor phase method

Broad-spectrum antibacterial activity of proteolytically stable self-assembled ag-hybrid peptide gels

Amphiphilic peptide-based supramolecular, noncytotoxic, stimuli-responsive hydrogels with antibacterial activity

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs

The effect of halogenation on the antimicrobial activity, antibiofilm activity, cytotoxicity and proteolytic stability of the antimicrobial peptide jelleine-I

Development of novel antimicrobial peptides derived from antilipopolysaccharide factor of the swimming crab, Portunus trituberculatus

De novo synthetic short antimicrobial peptides against cariogenic bacteria

Gene expression and in silico analysis of snakehead murrel interleukin 8 and antimicrobial activity of C-terminal derived peptide WS12

Novel bioactive peptides from PD-L1/2, a type 1 ribosome inactivating protein from phytolacca dioica L. Evaluation of their antimicrobial properties and anti-biofilm activities

Characterization of diverse antimicrobial peptides in skin secretions of chungan torrent frog amolops chunganensis

Pellino-1 derived cationic antimicrobial prawn peptide: bactericidal activity, toxicity and mode of action

A bovine myeloid antimicrobial peptide (BMAP-28) and its analogs kill pan-drug-resistant acinetobacter baumannii by interacting with outer membrane protein A (OmpA)

The activity and action mechanism of novel short selective LL-37-derived anticancer peptides against clinical isolates of Escherichia coli

Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy

Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures

Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles

Formation and properties of thin-walled phospholipid vesicles

Detergent-resistant, ceramideenriched domains in sphingomyelin/ceramide bilayers

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

Miscibility phase diagrams of giant vesicles containing sphingomyelin

Segregation of saturated chain lipids in pulmonary surfactant filmsand bilayers

Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions

Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles

To see or not to see: lateral organization of biological membranes and fluorescence microscopy

Giant vesicles, laurdan, and two-photon fluorescence microscopy: evidence of lipid lateral separation in bilayers

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Nord en, Membrane binding and translocation of cell-penetrating peptides

Single giant unilamellar vesicle method reveals effect of antimicrobial peptide magainin 2 on membrane permeability

Direct visualization of membrane leakage induced by the antibiotic peptides: maculatin, citropin, and aurein

Membrane interactions of antimicrobial peptides from Australian tree frogs

Stretch-Activated pore of the antimicrobial peptide

Single-vesicle detection and analysis of peptide-induced membrane permeabilization

Direct measurement of pore dynamics and leakage induced by a model antimicrobial peptide in single vesicles and cells

Continuous detection of entry of cell-penetrating peptide transporter 10 into single vesicles

Single-molecule fluorescence imaging of peptide binding to supported lipid bilayers

Probing membrane order and topography in supported lipid bilayers by combined polarized total internal reflection fluorescence-atomic force microscopy

Peptide-induced domain formation in supported lipid bilayers: direct evidence by combined atomic force and polarized total internal reflection fluorescence microscopy, Biophys

Combinatorial microscopy for the study of proteinmembrane interactions in supported lipid bilayers: order parameter measurements by combined polarized TIRFM/AFM

A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium Tuberculosis

Inflammatory and biocompatibility evaluation of antimicrobial peptide GL13K immobilized onto titanium by silanization

CyLoP-1: membrane-active peptide with cell-penetrating and antimicrobial properties

Membrane interactions of proline-rich antimicrobial peptide, chex1-arg20, multimers

Fungicidal mechanisms of the antimicrobial peptide Bac8c

The structure and behavior of the NA-CATH antimicrobial peptide with liposomes

Short, multiplestranded b-hairpin peptides have antimicrobial potency with high selectivity and salt resistance

Polymicrobial antibiofilm activity of the membranotropic peptide gH625 and its analogue

Scolopendin 2, a cationic antimicrobial peptide from centipede, and its membrane-active mechanism

Venom-derived peptide mastoparan-1 eradicates planktonic and biofilm-embedded methicillinresistant Staphylococcus aureus isolates

Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis

The antimicrobial peptide cathelicidin-BF could Be a potential therapeutic for Salmonella typhimurium infection

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

Characterization of chemoselective surface attachment of the cationic peptide melimine and its effects on antimicrobial activity

Identification of duck liver-expressed antimicrobial peptide 2 (LEAP-2) and characterization of its bactericidal activity

Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer

Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems

Peptide induced demixing in PG/PE lipid mixtures: a mechanism for the specificity of antimicrobial peptides towards bacterial membranes?

Headgroup structure and fatty acid chain length of the acidic phospholipids modulate the interaction of membrane mimetic vesicles with the antimicrobial peptide protegrin-1

Differential scanning microcalorimetry indicates that human defensin, hnp-2, interacts specifically with biomembrane mimetic systems

Interactions of the antimicrobial peptide Ac-FRWWHR-NH 2 with model membrane systems and bacterial cells

The interactions of antimicrobial peptides derived from lysozyme with model membrane systems

Interactions of tryptophan-rich cathelicidin antimicrobial peptides with model membranes studied by differential scanning calorimetry

Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles

Magainin 2 Amide Interaction with Lipid Membranes: Calorimetric Detection of Peptide Binding and Pore Formation

Binding of Antibacterial Magainin Peptides to Electrically Neutral Membranes: Thermodynamics and Structure

Membrane Binding and Pore Formation of the Antibacterial Peptide PGLa: Thermodynamic and Mechanistic Aspects

Thermodynamics of the a-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium11Edited by W

Thermodynamics of the coilea-helix transition of amphipathic peptides in a membrane environment: the role of vesicle curvature

Charge-Driven Interaction of Antimicrobial Peptide NK-2 with Phospholipid Membranes

Thermodynamic and Biophysical Analysis of the Membrane-Association of a Histidine-Rich Peptide with Efficient Antimicrobial and Transfection Activities

Interactions of the antimicrobial b-peptide b-17 with phospholipid vesicles differ from membrane interactions of magainins

Perturbation of the Hydrophobic Core of Lipid Bilayers by the Human Antimicrobial Peptide LL-37

Distinguishing between different pathways of bilayer disruption by the related antimicrobial peptides cecropin B, B1 and B3

Interaction of poly(l-lysines) with negatively charged membranes: an FT-IR and DSC study

Nonlamellar Phases Induced by the Interaction of Gramicidin S with Lipid Bilayers. A Possible Relationship to Membrane-Disrupting Activity

Solution Structure and Interaction of the Antimicrobial Polyphemusins with Lipid Membranes

MSI-78, an Analogue of the Magainin Antimicrobial Peptides, Disrupts Lipid Bilayer Structure via Positive Curvature Strain

SARS-CoV fusion peptides induce membrane surface ordering and curvature

Interaction of the Lipopeptide Biosurfactant Lichenysin with Phosphatidylcholine Model Membranes

Calorimetric and Spectroscopic Studies of the Effects of the Cell Penetrating Peptide Pep-1 and the Antimicrobial Peptide Combi-2 on Vesicles Mimicking Escherichia coli Membrane

Membrane interactions of proline-rich antimicrobial peptide, Chex1-Arg20, multimers

Resolving the structural interactions between antimicrobial peptides and lipid membranes using small-angle scattering methods: the case of indolicidin

Insights into the Mechanism of Action of the Two-Peptide Lantibiotic Lacticin 3147

Insight into the mechanism of action of temporin-SHa, a new broadspectrum antiparasitic and antibacterial agent

Role of non-electrostatic forces in antimicrobial potency of a dengue-virus derived fusion peptide VG16KRKP: Mechanistic insight into the interfacial peptide-lipid interactions

A thermodynamic signature of lipid segregation in biomembranes induced by a short peptide derived from glycoprotein gp36 of feline immunodeficiency virus

StructureeActivity Relationship of the Antimicrobial Peptide Gomesin: The Role of Peptide Hydrophobicity in Its Interaction with Model Membranes

riDOM, a cell penetrating peptide. Interaction with phospholipid bilayers

S.M and B.B acknowledge Direcci on General Asuntos del Personal Acad emico for postdoctoral fellowship. Carlos Munoz-Garay acknowledges PAPIIT/UNAM project IN209318 financing. This work was also supported by Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED) (219RT0573). Red tem atica en salud. "Desarrollo de p eptidos antivirales y antimicrobianos para cepas multi-resistentes". Special thanks to the anonymous reviewers for carefully reading our manscript and for their valuable observations and opinions that greatly improved this article.