General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides.

Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by ³¹P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

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

Dynamic structures of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers induced in oriented lipid membranes, which are interacting with membrane-acting antimicrobial peptides (AMPs), magainin-2 and aurein-3.3, were explored by ³¹P and ²H solid-state NMR (ssNMR) spectroscopy. Various types of phospholipid systems, such as POPC-d₃₁, POPC-d₃₁/POPG, and POPC-d₃₁/cholesterol, were investigated to understand the membrane disruption mechanisms of magainin-2 and aurein-3.3 peptides at various peptide-to-lipid (P:L) ratios. The experimental lineshapes of anisotropic ³¹P and ²H ssNMR spectra measured on these peptide-lipid systems were simulated reasonably well by assuming the presence of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers, in membranes. Furthermore, the observed decrease in the anisotropic frequency span of either ³¹P or ²H ssNMR spectra of oriented lipid bilayers, particularly when anionic POPG lipids are interacting with AMPs at high P:L ratios, can directly be explained by a thinned membrane surface model with fast lateral diffusive motions of lipids. The spectral analysis protocol we developed enables extraction of the lateral diffusion coefficients of lipids distributed on the curved surfaces of pores and thinned bilayers on a few nanometers scale.     

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: Insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. ³¹P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, ²H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.     

Permeation of membranes by the neutral form of amino acids and peptides: Relevance to the origin of peptide translocation

The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10⁵ slower than facilitated inward transport across biological membranes. This suggests that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10¹⁰ times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g., for certain signal sequences, toxins and thylakoid proteins) in vivo.Abbreviations LUV large unilamellar vesicle - pH transmembrane pH gradient - PAH polyaromatic hydrocarbon Correspondence to: A.C. Chakrabarti     

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence–function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX₁HNGRHX₂C, cCX₁HNGRAX₂C, and cCX₁ANGRHX₂C) are related with the position of His residues and cationic amino acids.     

3D-Structure of the interior fusion peptide of HGV/GBV-C by 1H NMR, CD and molecular dynamics studies

In this work, we present a structural characterization of the putative fusion peptide E2(279-298) corresponding to the E2 envelope protein of the HGV/GBV-C virus by (1)H NMR, CD and MD studies performed in H(2)O/TFE and in lipid model membranes. The peptide is largely unstructured in water, whereas in H(2)O/TFE and in model membranes it adopts an helical structure (approximately 65-70%). The partitioning free energy DeltaG ranges from -6 to -7.5 kcal mol(-1). OCD measurements on peptide-containing hydrated and oriented lipid multilayers showed that the peptide adopts a predominantly surface orientation. The (1)H NMR data (observed NOEs, deuterium exchange rates, Halpha chemical shift index and vicinal coupling constants) and the molecular dynamics calculations support the conclusions that the peptide adopts a stable helix in the C-terminal 9-18 residues slightly inserted into the lipid bilayer and a major mobility in the amino terminus of the sequence (1-8 residues).     

Effect of structural parameters of peptides on dimer formation and highly oxidized side products in the oxidation of thiols of linear analogues of human β‐defensin 3 by DMSO

The purpose of this study was to examine the effects of structural parameters of peptides on their oxidation by DMSO, including location of cysteine, effect of adjunct group participation, molecular hydrophobicity, steric hindrance or the accessibility of thiol group and peptide conformation, on oxidation rates, dimer formation and associated side products. We designed and synthesized two series of linear cysteine‐containing analogues of human β‐defensin 3 (the C1‐peptides with cysteine at the N‐terminus residue 1, the C29‐peptides with cysteine located at residue 29 in the centre of peptide), which were used for preparation of disulphide‐linked homodimers. HPLC–ESI–MS was used to monitor the oxidation process and to characterize the molecular weights of dimers and side products of high oxidation. The formations of dimers and side products were dependent on the position of cysteines. Hydrophobicity generally rendered the thiol groups less accessible and hence exposed them to slow oxidation to form dimers (or even fail to form dimers during the timescale of observation). Molecular dynamics simulations showed that the exposure of cysteines (and sulphurs) of the C1‐peptides was much larger than for the C29‐peptides. The larger hydrophobic side chains tended to enable clustering of the side chains that sequester cysteine, particularly in the C29‐peptides, which provided a molecular explanation for the observed trends in oxidation rates. Together with molecular modelling, we propose a reaction mechanism to elucidate the oxidation results of these peptides. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.