Antimicrobial peptides in toroidal and cylindrical pores

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize biological membranes. Their mechanism of action is still not well understood. Here we investigate the preference of alamethicin and melittin for pores of different shapes, using molecular dynamics (MD) simulations of the peptides in pre-formed toroidal and cylindrical pores. When an alamethicin hexamer is initially embedded in a cylindrical pore, at the end of the simulation the pore remains cylindrical or closes if glutamines in the N-termini are not located within the pore. On the other hand, when a melittin tetramer is embedded in toroidal pore or in a cylindrical pore, at the end of the simulation the pore is lined both with peptides and lipid headgroups, and, thus, can be classified as a toroidal pore. These observations agree with the prevailing views that alamethicin forms barrel-stave pores whereas melittin forms toroidal pores. Both alamethicin and melittin form amphiphilic helices in the presence of membranes, but their net charge differs; at pH ∼ 7, the net charge of alamethicin is − 1 whereas that of melittin is + 5. This gives rise to stronger electrostatic interactions of melittin with membranes than those of alamethicin. The melittin tetramer interacts more strongly with lipids in the toroidal pore than in the cylindrical one, due to more favorable electrostatic interactions.     

On the mechanism of pore formation by melittin

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.     

The Structure of a Melittin-Stabilized Pore

Melittin has been reported to form toroidal pores under certain conditions, but the atomic-resolution structure of these pores is unknown. A 9-μs all-atom molecular-dynamics simulation starting from a closely packed transmembrane melittin tetramer in DMPC shows formation of a toroidal pore after 1 μs. The pore remains stable with a roughly constant radius for the rest of the simulation. Surprisingly, one or two melittin monomers frequently transition between transmembrane and surface states. All four peptides are largely helical. A simulation in a DMPC/DMPG membrane did not lead to a stable pore, consistent with the experimentally observed lower activity of melittin on anionic membranes. The picture that emerges from this work is rather close to the classical toroidal pore, but more dynamic with respect to the configuration of the peptides.     

What Makes a Good Pore Former: A Study of Synthetic Melittin Derivatives

Pore formation by membrane-active peptides, naturally encountered in innate immunity and infection, could have important medical and technological applications. Recently, the well-studied lytic peptide melittin has formed the basis for the development of combinatorial libraries from which potent pore-forming peptides have been derived, optimized to work under different conditions. We investigate three such peptides, macrolittin70, which is most active at neutral pH; pHD15, which is active only at low pH; and MelP5_Δ6, which was rationally designed to be active at low pH but formed only small pores. There are three, six, and six acidic residues in macrolittin70, pHD15, and MelP5_Δ6, respectively. We perform multi-microsecond simulations in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of hexamers of these peptides starting from transmembrane orientations at neutral pH (all residues at standard protonation), low pH (acidic residues and His protonated), and highly acidic environments in which C-termini are also protonated. Previous simulations of the parent peptides melittin and MelP5 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are repeated in POPC. We find that the most potent pore-forming peptides exhibit strong interpeptide interactions, including salt bridges, H-bonds, and polar interactions. Protonation of the C-terminus promotes helicity and pore size. The proximity of the peptides allows fewer lipid headgroups to line the pores than in previous simulations, making the pores intermediate between barrel stave and toroidal. Based on these structures and geometrical arguments, we attempt to rationalize the factors that under different conditions can increase or decrease pore stability and propose mutations that could be tested experimentally.     

Charge distribution and imperfect amphipathicity affect pore formation by antimicrobial peptides

Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analog MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136ns to 216ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melittin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its configuration diverges from that of melittin and it is probably metastable.     

Organizations of melittin peptides after spontaneous penetration into cell membranes

The antimicrobial peptide, melittin, is a potential next-generation antibiotic because melittin can spontaneously form pores in bacterial cell membranes and cause cytoplasm leakage. However, the organizations of melittin peptides in cell membranes remain elusive, which impedes the understanding of the poration mechanism. In this work, we use coarse-grained and all-atom molecular dynamics (MD) simulations to investigate the organizations of melittin peptides during and after spontaneous penetration into DPPC/POPG lipid bilayers. We find that the peptides in lipid bilayers adopt either a transmembrane conformation or a U-shaped conformation, which are referred to as T- and U-peptides, respectively. Several U-peptides and/or T-peptides aggregate to form stable pores. We analyze a T-pore consisting of four T-peptides and a U-pore consisting of three U-peptides and one T-peptide. In both pores, peptides are organized in a manner such that polar residues face inward and hydrophobic residues face outward, which stabilizes the pores and produces water channels. Compared with the U-pore, the T-pore has lower energy, larger pore diameter, and higher permeability. However, the T-pore occurs less frequently than the U-pore in our simulations, probably because the formation of the T-pore is kinetically slower than the U-pore. The stability and permeability of both pores are confirmed by 300 ns all-atom MD simulations. The peptide organizations obtained in this work should deepen the understanding of the stability, poration mechanism, and permeability of melittin, and facilitate the optimization of melittin to enhance the antibacterial ability.     

Toroidal pores formed by antimicrobial peptides show significant disorder

A large variety of antimicrobial peptides have been shown to act, at least in vitro, by poration of the lipid membrane. The nanometre size of these pores, however, complicates their structural characterization by experimental techniques. Here we use molecular dynamics simulations, to study the interaction of a specific class of antimicrobial peptides, melittin, with a dipalmitoylphosphatidylcholine bilayer in atomic detail. We show that transmembrane pores spontaneously form above a critical peptide to lipid ratio. The lipid molecules bend inwards to form a toroidally shaped pore but with only one or two peptides lining the pore. This is in strong contrast to the traditional models of toroidal pores in which the peptides are assumed to adopt a transmembrane orientation. We find that peptide aggregation, either prior or after binding to the membrane surface, is a prerequisite to pore formation. The presence of a stable helical secondary structure of the peptide, however is not. Furthermore, results obtained with modified peptides point to the importance of electrostatic interactions in the poration process. Removing the charges of the basic amino-acid residues of melittin prevents pore formation. It was also found that in the absence of counter ions pores not only form more rapidly but lead to membrane rupture. The rupture process occurs via a novel recursive poration pathway, which we coin the Droste mechanism.     

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

Transmembrane pores induced by amphiphilic peptides, including melittin, are often modeled with the barrel-stave model after the alamethicin pore. We examine this assumption on melittin by using two methods, oriented circular dichroism (OCD) for detecting the orientation of melittin helix and neutron scattering for detecting transmembrane pores. OCD spectra of melittin were systematically measured. Melittin can orient either perpendicularly or parallel to a lipid bilayer, depending on the physical condition and the composition of the bilayer. Transmembrane pores were detected when the helices oriented perpendicularly to the plane of the bilayers, not when the helices oriented parallel to the bilayers. The evidence that led to the barrel-stave model for alamethicin and that to the toroidal model for magainin were reviewed. The properties of melittin pores are closely similar to that of magainin but unlike that of alamethicin. We conclude that, among naturally produced peptides that we have investigated, only alamethicin conforms to the barrel-stave model. Other peptides, including magainins, melittin and protegrins, all appear to induce transmembrane pores that conform to the toroidal model in which the lipid monolayer bends continuously through the pore so that the water core is lined by both the peptides and the lipid headgroups.     

Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

Exploring peptide membrane interaction using surface plasmon resonance: differentiation between pore formation versus membrane disruption by lytic peptides

Lytic peptides comprise a large group of membrane-active peptides used in the defensive and offensive systems of all organisms. Differentiating between their modes of interaction with membranes is crucial for understanding how these peptides select their target cells. Here we utilized SPR to study the interaction between lytic peptides and lipid bilayers (L1 sensor chip). Using studies also on hybrid monolayers (HPA sensor chip) revealed that SPR is a powerful tool for obtaining a real-time monitoring of the steps involved in the mode of action of membrane-active peptides, some of which previously could not be detected directly by other techniques and reported here for the first time. We investigated the mode of action of peptides that represent two major families: (i) the bee venom, melittin, as a model of a non-cell-selective peptide that forms transmembrane pores and (ii) magainin and a diastereomer of melittin (four amino acids were replaced by their D enantiomers), as models of bacteria-selective non-pore-forming peptides. Fitting the SPR data to different interaction models allows differentiating between two major steps: membrane binding and membrane insertion. Melittin binds to PC/cholesterol approximately 450-fold better than its diastereomer and magainin, mainly because it is inserted into the inner leaflet (2/3 of the binding energy), whereas the other two are not. In contrast, there is only a slight difference in the binding of all the peptides to negatively charged PE/PG mono- and bilayer membranes (in the first and second steps), indicating that the inner leaflet contributes only slightly to their binding to PE/PG bilayers. Furthermore, the 100-fold stronger binding of the cell-selective peptides to PE/PG as compared with PC/cholesterol resulted only from electrostatic attraction to the negatively charged headgroups of the outer leaflet. These results clearly differentiate between the two general mechanisms: pore formation by melittin only in zwitterionic membranes and a detergent-like effect (carpet mechanism) for all the peptides in negatively charged membranes, in agreement with their biological function.     

Interaction of linear cationic peptides with phospholipid membranes and polymers of sialic acid

Polysialic acid (PSA) is a natural anionic polymer typically occurring on the outer surface of cell membranes. PSA is involved in cell signaling and intermolecular interactions with proteins and peptides. The antimicrobial potential of peptides is usually evaluated in model membranes consisting of lipid bilayers but devoid of either PSA or its analogs. The goal of this work was to investigate the possible effect of PSA on the structure of melittin (Mlt) and latarcins Ltc1K, Ltc2a, and the activity of these peptides with respect to model membranes. These peptides are linear cationic ones derived from the venom of bee (Mlt) and spider (both latarcins). The length of each of the peptides is 26 amino acid residues, and they all have antimicrobial activity. However, they differ with respect to conformational mobility, hydrophobic characteristics, and overall charge. In this work, using circular dichroism spectroscopy, we show that the peptides adopt an α-helical conformation upon interaction with either PSA or phospholipid liposomes formed of either zwitterionic or anionic phospholipids or their mixtures. The extent of helicity depends on the amino acid sequence and properties of the medium. Based on small angle X-ray scattering data and the analysis of the fluorescence spectrum of the Trp residue in Mlt, we conclude that the peptide forms an oligomeric complex consisting of α-helical Mlt and several PSA molecules. Both latarcins, unlike Mlt, the most hydrophobic of the peptides, interact weakly with zwitterionic liposomes. However, they bind anionic liposomes or those composed of anionic/zwitterionic lipid mixtures. Latarcin Ltc1K forms associates on liposomes composed of zwitterionic/anionic lipid mixture. The structure of the peptide associates is either disordered or of β-sheet conformation. In all other cases the studied peptides adopt predominately α-helical conformation. In addition, we demonstrate that PSA inhibits membranolytic activity of Mlt and latarcin Ltc1K. These data suggest that the peptides, due to their high conformational lability, can vary structural and amphiphilic properties in the presence of PSA. As a result, various scenarios of the interaction of the peptides with membranes, whose surface is abundant with anionic polysaccharides, can take place. This can account for difficulties in understanding the structure-functional relationships in interactions of linear cationic peptides with biological membranes.     

The role of electrostatic interactions in the membrane binding of melittin

The binding of melittin and the C-terminally truncated analogue of melittin (21Q) to a range of phospholipid bilayers was studied using surface plasmon resonance (SPR). The phospholipid model membranes included zwitterionic dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylethanolamine (DMPE), together with mixtures DMPC/dimyristylphosphatidylglycerol (DMPG), DMPC/DMPG/cholesterol and DMPE/DMPG. Melittin bound rapidly to all membrane mixtures, whereas 21Q, which has a reduced charge, bound much more slowly on the DMPC and DMPC/DMPG mixtures reflecting the role of the initial electrostatic interaction. The loss of the cationic residues also significantly decreased the binding of 21Q with DMPC/DMPG/Cholesterol, DMPE and DMPE/DMPG. The role of electrostatics was also highlighted with NaCl in the buffer, which affected the way melittin bound to the different membranes, causing a more uniform, concentration dependant increase in response. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrate that the positively charged residues at the C-terminus of melittin play an essential role in membrane binding, that modulation of peptide charge influences selectivity of binding to different phospholipids and that manipulation of the cationic regions of antimicrobial peptides can be used to modulate membrane selectivity.     

Effects of arginine density on the membrane-bound structure of a cationic antimicrobial peptide from solid-state NMR

Solid-state NMR spectroscopy is used to determine the membrane-bound topological structure of a cationic β-hairpin antimicrobial peptide in which the number of Arg residues has been halved. The parent peptide, PG-1, was previously found to form transmembrane β-barrels in anionic membranes where the Arg residues complex with the lipid phosphate groups to cause toroidal pore defects in the membrane. In comparison, the charge-attenuated and less active mutant studied here forms β-sheets that lie on the surface of the zwitterionic membrane and only partially insert into the anionic membrane. The mutant also exhibits much looser contact with the lipid headgroups. These results indicate that transmembrane insertion and tight Arg-phosphate association are two important elements for strong antimicrobial activities of this class of peptides. Comparison with other β-hairpin antimicrobial peptides studied so far further suggests a relative potency scale for the various mechanisms of action for the β-sheet family of antimicrobial peptides. The transmembrane insertion-toroidal pore mechanism is the most potent in disrupting the lipid bilayer, followed by the large-amplitude in-plane motional mechanism. The carpet model, where peptides aggregate on the membrane surface to cause lateral expansion and eventual micellization of the membrane, is a weaker mechanism of action.     

Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides

In this work, we sought to rationally design membrane-active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane-active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6, by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macromolecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH sensitivity into its sequence. We conclude that the properties of pHlip, GALA, and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane-active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field.     

Effect of micellar charge on the conformation and dynamics of melittin

Electrostatic interactions play a crucial role in modulating and stabilizing molecular interactions in membranes and membrane-mimetic systems such as micelles. We have monitored the change in the conformation and dynamics of the cationic hemolytic peptide melittin bound to micelles of various charge types, utilizing fluorescence and circular dichroism (CD) spectroscopy. The sole tryptophan of melittin displays a red-edge excitation shift (REES) of 3-6 nm when bound to anionic, nonionic, and zwitterionic micelles. This suggests that melittin is localized in a restricted environment, probably in the interfacial region of the micelles, and this region offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state tryptophan in melittin. Further, the rotational mobility of melittin is considerably reduced in these micelles and is found to be dependent on the surface charge of micelles. Interestingly, our results show that melittin does not partition into cetyltrimethylammonium bromide (CTAB) micelles owing to electrostatic repulsion between melittin and CTAB micelles, both of which carry a positive charge. In addition, the fluorescence lifetime of melittin is modulated in micelles of different charge types. The lowest mean fluorescence lifetime is observed in the case of melittin bound to anionic sodium dodecyl sulfate (SDS) micelles. CD spectroscopy shows that micelles induce significant helicity to melittin, with maximum helicity being induced in the case of melittin bound to SDS micelles. Fluorescence quenching measurements using the neutral aqueous quencher acrylamide show differential accessibility of melittin in various types of micelles. Taken together, our results show that micellar surface charge can modulate the conformation and dynamics of melittin. These results could be relevant to understanding the role of the surface charge of membranes in the interaction of membrane-active, amphiphilic peptides with membranes.     

Pore Structure and Synergy in Antimicrobial Peptides of the Magainin Family

Magainin 2 and PGLa are among the best-studied cationic antimicrobial peptides. They bind preferentially to negatively charged membranes and apparently cause their disruption by the formation of transmembrane pores, whose detailed structure is still unclear. Here we report the results of 5–9 μs all-atom molecular dynamics simulations starting from tetrameric transmembrane helical bundles of these two peptides, as well as their stoichiometric mixture, and the analog MG-H2 in DMPC or 3:1 DMPC/DMPG membranes. The simulations produce pore structures that appear converged, although some effect of the starting peptide arrangement (parallel vs. antiparallel) is still observed on this timescale. The peptides remain mostly helical and adopt tilted orientations. The calculated tilt angles for PGLa are in excellent agreement with recent solid state NMR experiments. The antiparallel dimer structure in the magainin 2 simulations resembles previously determined NMR and crystal structures. More transmembrane orientations and a larger and more ordered pore are seen in the 1:1 heterotetramer with an antiparallel helix arrangement. Insights into the mechanism of synergy between these two peptides are obtained via implicit solvent modeling of homo- and heterodimers and analysis of interactions in the atomistic simulations. This analysis suggests stronger pairwise interactions in the heterodimer than in the two homodimers.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

Free energy analysis of membrane pore formation process in the presence of multiple melittin peptides

Understanding the molecular mechanism underlying pore formation in lipid membranes by antimicrobial peptides is of great importance in biological sciences as well as in drug design applications. Melittin has been widely studied as a pore forming peptide, though the molecular mechanism for pore formation is still illusive. We examined the free energy barrier for the creation of a pore in lipid membranes with and without multiple melittin peptides. It was found that six melittin peptides significantly stabilized a pore, though a small barrier (a few k_BT) for the formation still existed. With five melittin peptides or fewer, the pore formation barrier was much higher, though the established pore was in a local energy minimum. Although seven melittins effectively reduced the free energy barrier, a single melittin peptide left the pore after a long time MD simulation probably because of the overcrowded environment around the bilayer pore. Thus, it is highly selective for the number of melittin peptides to stabilize the membrane pore, as was also suggested by the line tension evaluations. The free energy cost required to insert a single melittin into the membrane is too high to explain the one-by-one insertion mechanism for pore formation, which also supports the collective melittin mechanism for pore formation.     

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.     

Determining the mechanism of membrane permeabilizing peptides: Identification of potent, equilibrium pore-formers

To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles, and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.