Binding and insertion of alpha-helical anti-microbial peptides in POPC bilayers studied by molecular dynamics simulations

We have performed molecular dynamics simulations of the interactions of two alpha-helical anti-microbial peptides, magainin2 and its synthetic analog of MSI-78, with palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers. We used various initial positions and orientations of the peptide with respect to the lipid bilayer, including a surface-bound state parallel to the interface, a trans-membrane state, and a partially inserted state. Our 20 ns long simulations show that both magainin2 and MSI-78 are most stable in the lipid environment, with the peptide destabilized to different extents in both aqueous and lipid/water interfacial environments. We found that there are strong specific interactions between the lysine residues of the peptides and the lipid head-group regions. MSI-78, owing to its large number of lysines, shows better binding characteristics and overall stability when compared to magainin2. We also find that both peptides destabilize the bilayer environment, as observed by the increase in lipid tail disorder and the induction of local curvature on the lipid head-groups by the peptides. From all the simulations, we conclude that the hydrogen bonding interactions between the lysines of the peptides and the oxygens of the polar lipid head-groups are the strongest and determine the overall peptide binding characteristics to the lipids.     

Effect of salt on the interactions of antimicrobial peptides with zwitterionic lipid bilayers

The effect of salt on the binding of the antimicrobial peptide magainin to POPC lipid bilayers is studied by 40-50 ns molecular dynamics simulations of a POPC bilayer in the presence of different concentrations of Na+ and Cl- ions, corresponding to effective concentrations of 0, 100, 150, 200, 250 and 300 millimolar NaCl, with and without a single molecule of antimicrobial peptide magainin. Simulations without magainin showed that increasing salt concentration leads to the decrease in the area per lipid, a decrease in the head group tilt of the lipids, as well as increased order of lipid tails, in agreement with other recent simulations. Simulations with magainin show that peptide binding to the lipids is stronger at lower concentrations of salt. The peptides disorder the lipids in their immediate vicinity, but this effect is diminished as the salt concentration increases. Our studies indicate that while 50 ns simulations give information on peptide hydrogen bonding and lipid tail ordering that is insensitive to the initial peptide orientation, this run time is not sufficient to equilibrate the peptide position and orientation within the bilayer.     

Effect of salt on the interactions of antimicrobial peptides with zwitterionic lipid bilayers

The effect of salt on the binding of the antimicrobial peptide magainin to POPC lipid bilayers is studied by 40-50 ns molecular dynamics simulations of a POPC bilayer in the presence of different concentrations of Na+ and Cl− ions, corresponding to effective concentrations of 0, 100, 150, 200, 250 and 300 millimolar NaCl, with and without a single molecule of antimicrobial peptide magainin. Simulations without magainin showed that increasing salt concentration leads to the decrease in the area per lipid, a decrease in the head group tilt of the lipids, as well as increased order of lipid tails, in agreement with other recent simulations. Simulations with magainin show that peptide binding to the lipids is stronger at lower concentrations of salt. The peptides disorder the lipids in their immediate vicinity, but this effect is diminished as the salt concentration increases. Our studies indicate that while 50 ns simulations give information on peptide hydrogen bonding and lipid tail ordering that is insensitive to the initial peptide orientation, this run time is not sufficient to equilibrate the peptide position and orientation within the bilayer.     

Analysis of antimicrobial peptide interactions with hybrid bilayer membrane systems using surface plasmon resonance

The lipid binding behaviour of the antimicrobial peptides magainin 1, melittin and the C-terminally truncated analogue of melittin (21Q) was studied with a hybrid bilayer membrane system using surface plasmon resonance. In particular, the hydrophobic association chip was used which is composed of long chain alkanethiol molecules upon which liposomes adsorb spontaneously to create a hybrid bilayer membrane surface. Multiple sets of sensorgrams with different peptide concentrations were generated. Linearisation analysis and curve fitting using numerical integration analysis were performed to derive estimates for the association (k(a)) and dissociation (k(d)) rate constants. The results demonstrated that magainin 1 preferentially interacted with negatively charged dimyristoyl-L-alpha-phosphatidyl-DL-glycerol (DMPG), while melittin interacted with both zwitterionic dimyristoyl-L-alpha-phosphatidylcholine and anionic DMPG. In contrast, the C-terminally truncated melittin analogue, 21Q, exhibited lower binding affinity for both lipids, showing that the positively charged C-terminus of melittin greatly influences its membrane binding properties. Furthermore the results also demonstrated that these antimicrobial peptides bind to the lipids initially via electrostatic interactions which then enhances the subsequent hydrophobic binding. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high alpha-helicity was associated with high binding affinity. Overall, the results demonstrated that biosensor technology provides a new experimental approach to the study of peptide-membrane interactions through the rapid determination of the binding affinity of bioactive peptides for phospholipids.     

Biological activity and structural aspects of PGLa interaction with membrane mimetic systems

Peptidyl-glycine-leucine-carboxyamide (PGLa), isolated from granular skin glands of Xenopus laevis, is practically devoid of secondary structure in aqueous solution and in the presence of zwitterionic phospholipids, when added exogenously, but adopts an α-helix in the presence of anionic lipids. The peptide was shown to exhibit antifungal activity and to have antimicrobial activity towards both Gram-negative and Gram-positive bacteria. As a broad variety of peptides is found in the secretions of amphibian skin combinatorial treatment of PGLa and magainin 2 was studied showing enhanced activity by a heterodimer formation. Thus production of mutually recognizing peptides seems to be an effective way in nature to increase selective membrane activity. Biophysical studies on membrane mimics demonstrated that PGLa can discriminate between different lipid species, preferentially interacting with negatively charged lipids, which are major components of bacterial but not mammalian cell membranes. This emphasizes the role of electrostatic interactions as a major determinant to trigger the affinity of antimicrobial peptides towards bacterial membranes. PGLa induced the formation of a quasi-interdigitated phase in phosphatidylglycerol bilayers below their chain melting transition, which is due to the creation of voids below the peptide being aligned parallel to the membrane surface. In the fluid phase of phosphatidylglycerol the peptide inserts perpendicularly into the bilayer above a threshold concentration, which results in a hydrophobic mismatch of the peptide length and bilayer core for lipids ≤ C16. This mismatch is compensated by stretching of the acyl chains and in turn thickening of the bilayer demonstrating that membrane thinning cannot be taken generally as the hallmark of pore formation by antimicrobial peptides. Furthermore, PGLa was shown to affect membrane curvature strain of phosphatidylethanolamine, another main lipid component of bacterial membranes, where a cubic phase coexists with the fluid bilayer phase. Investigations on living Escherichia coli showed distinct changes in cell envelope morphology, when treated with the peptide. In a first stage loss of surface stiffness and consequently of topographic features was observed, followed in a second stage by permeabilization of the outer membrane and rupture of the inner (cytoplasmic) membrane supposedly by the mechanism(s) derived from model studies.     

Structural contributions to the intracellular targeting strategies of antimicrobial peptides

The interactions of cationic amphipathic antimicrobial peptides (AMPs) with anionic biological membranes have been the focus of much research aimed at improving the activity of such compounds in the search for therapeutic leads. However, many of these peptides are thought to have other polyanions, such as DNA or RNA, as their ultimate target. Here a combination of fluorescence and circular dichroism (CD) spectroscopies has been used to assess the structural properties of amidated versions of buforin II, pleurocidin and magainin 2 that support their varying abilities to translocate through bacterial membranes and bind to double stranded DNA. Unlike magainin 2 amide, a prototypical membrane disruptive AMP, buforin II amide adopts a poorly helical structure in membranes closely mimicking the composition of Gram negative bacteria, such as Escherichia coli, and binds to a short duplex DNA sequence with high affinity, ultimately forming peptide-DNA condensates. The binding affinities of the peptides to duplex DNA are shown to be related to the structural changes that they induce. Furthermore, CD also reveals the conformation of the bound peptide buforin II amide. In contrast with a synthetic peptide, designed to adopt a perfect amphipathic α-helix, buforin II amide adopts an extended or polyproline II conformation when bound to DNA. These results show that an α-helix structure is not required for the DNA binding and condensation activity of buforin II amide.     

Diffusion as a probe of peptide-induced membrane domain formation

Recently, we have shown that association with an antimicrobial peptide (AMP) can drastically alter the diffusion behavior of the constituent lipids in model membranes (Biochemistry 49, 4672-4678). In particular, we found that the diffusion time of a tracer fluorescent lipid through a confocal volume measured via fluorescence correlation spectroscopy (FCS) is distributed over a wide range of time scales, indicating the formation of stable and/or transient membrane species that have different mobilities. A simple estimate, however, suggested that the slow diffusing species are too large to be attributed to AMP oligomers or pores that are tightly bound to a small number of lipids. Thus, we tentatively ascribed them to membrane domains and/or clusters that possess distinctively different diffusion properties. In order to further substantiate our previous conjecture, herein we study the diffusion behavior of the membrane-bound peptide molecules using the same AMPs and model membranes. Our results show, in contrast to our previous findings, that the diffusion times of the membrane-bound peptides exhibit a much narrower distribution that is more similar to that of the lipids in peptide-free membranes. Thus, taken together, these results indicate that while AMP molecules prompt domain formation in membranes, they are not tightly associated with the lipid domains thus formed. Instead, they are likely located at the boundary regions separating various domains and acting as mobile fences.     

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

Magainin 2 is a 23-residue peptide that forms an amphipathic alpha-helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One- and two-dimensional solid-state 15N NMR spectra of specifically 15N-labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is alpha-helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.     

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.     

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

We have investigated the structure of lipid bilayers containing varied molar ratios of different lipids and the antimicrobial peptides magainin and alamethicin. For this structural study, we have used x-ray reflectivity on highly aligned solid-supported multilamellar lipid membranes. The reflectivity curves have been analyzed by semi-kinematical reflectivity theory modeling the bilayer density profile rho(z). Model simulations of the reflectivity curves cover a large range of vertical momentum transfer q(z), and yield excellent agreement between data and theory. The structural changes observed as a function of the molar peptide/lipid concentration P/L are discussed in a comparative way.     

The peptide antibiotic clavanin A interacts strongly and specifically with lipid bilayers

In this study the interaction of the antimicrobial peptide clavanin A with phosphatidylcholine bilayers is investigated by DSC, NMR, and AFM techniques. It is shown that the peptide interacts strongly and specifically with the lipids, resulting in increased order-disorder phase transition temperatures, phase separation, altered acyl chain and headgroup packing, and a drastically changed surface morphology of the bilayer. These results are interpreted in terms of clavanin-specific interactions with lipids and are discussed in the light of the different mechanisms by which clavanin A can destroy the barrier function of biological membranes.     

Amphipathic Helical Cationic Antimicrobial Peptides Promote Rapid Formation of Crystalline States in the Presence of Phosphatidylglycerol: Lipid Clustering in Anionic Membranes

Five AHCAPs exhibiting a broad-spectrum of antimicrobial activity, were examined with regard to their action in lipid mixtures with two anionic lipids, PG and CL. We find that all of the peptides studied were capable of promoting the formation of crystalline phases of DMPG in mixtures of DMPG and CL, without prior incubation at low temperatures. This property is indicative of the ability of these peptides to cluster CL away from DMPG. In contrast, the well studied antimicrobial cationic peptide magainin 2 does not cluster anionic lipids. We ascribe the lower anionic lipid clustering ability of magainin to its low density of positive charges compared with the five other AHCAPs used in this work. The peptide MSI-1254 was particularly potent in segregating these two anionic lipids. Consequently, clusters enriched in DMPG appear in a lipid mixture with CL. These can rapidly form higher temperature crystalline phases because of the increased permeability of the bilayer caused by the AHCAPs. The polyaminoacids, poly-L-Lysine and poly-l-arginine are also very effective in causing this segregation. Thus, the clustering of anionic lipids by AHCAPs is not confined only to mixtures of anionic with zwitterionic lipids, but it extends to mixtures containing different anionic headgroups. The resulting effects, however, have different consequences to the biological activity. This finding broadens the scope for which an AHCAP agent will cluster lipids in a membrane.     

Membrane insertion and bilayer perturbation by antimicrobial peptide CM15

Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2-9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an alpha-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2-3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the (31)P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. (2)H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. (31)P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, (31)P and (2)H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol

Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.     

Is lipid bilayer binding a common property of inhibitor cysteine knot ion-channel blockers?

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (DeltaG) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of DeltaG dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep DeltaG dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed DeltaG shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.     

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous diffusion behavior in the probe population.     

Interaction Between Amyloid-β (1-42) Peptide and Phospholipid Bilayers: A Molecular Dynamics Study

The amyloid-β (Aβ) peptide is a key aggregate species in Alzheimer's disease. Although important aspects of Aβ peptide aggregation are understood, the initial stage of aggregation from monomer to oligomer is still not clear. One potential mediator of this early aggregation process is interactions of Aβ with anionic cell membranes. We used unconstrained and umbrella sampling molecular dynamics simulations to investigate interactions between the 42-amino acid Aβ peptide and model bilayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) lipids and anionic dioleoylphosphatidylserine (DOPS) lipids. Using these methods, we determined that Aβ is attracted to the surface of DPPC and DOPS bilayers over the small length scales used in these simulations. We also found supporting evidence that the charge on both the bilayer surface and the peptide affects the free energy of binding of the peptide to the bilayer surface and the distribution of the peptide on the bilayer surface. Our work demonstrates that interactions between the Aβ peptide and lipid bilayer promotes a peptide distribution on the bilayer surface that is prone to peptide-peptide interactions, which can influence the propensity of Aβ to aggregate into higher-order structures.     

Position-dependent hydrophobicity of the antimicrobial magainin peptide affects the mode of peptide-lipid interactions and selective toxicity

Cationic antimicrobial peptides are promising candidates as novel antibiotics of clinical usefulness. Magainin 2, a representative antimicrobial peptide isolated from the skin of the African clawed frog Xenopus leavis, electrostatically recognizes anionic lipids that are abundant in bacterial membranes, forming a peptide-lipid supramolecular complex pore, whereas the peptide does not effectively bind to zwitterionic phospholipids constituting the outer leaflets of mammalian cell membranes because of the low hydrophobicity of the peptide [Matsuzaki, K. (1999) Biochim. Biophys. Acta 1462, 1-10]. In this study, two magainin analogues with enhanced hydrophobicity, MG-H1 (GIKKFLHIIWKFIKAFVGEIMNS) and MG-H2 (IIKKFLHSIWKFGKAFVGEIMNI), with identical amino acid compositions were designed and interactions with lipid bilayers and biological activities were examined in comparison with those of MG (GIGKWLHSAKKFGKAFVGEIMNS = F5W-magainin 2). The apparent hydrophobicities and hydrophobic moments of MG-H1 and MG-H2, conventionally calculated assuming that all residues are involved in helix formation, were almost the same. MG-H2 behaved like MG except for greatly enhanced activity against zwitterionic membranes and erythrocytes. In contrast, despite a very similar calculated hydrophobicity, the observed hydrophobicity of MG-H1 was larger than that of MG-H2 because of a tendency toward helix fraying near the termini. Therefore, the physicochemical parameters of only the helical portion should be considered in characterizing peptide-lipid interactions, although this point was overlooked in most studies. Moreover, MG-H1 induced aggregation and/or fusion of negatively charged membranes. Furthermore, the peptide hydrophobicity was found to affect pore formation rate, pore size, and pore stability. These observations demonstrate that the hydrophobicity of the peptide also controls the mode of action and is dependent on the position of the hydrophobic amino acids in the peptide sequence.     

Membrane binding mechanism of an RNA virus-capping enzyme

The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation. NMR structure shows that the alpha-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.