Conformation and interaction of the cyclic cationic antimicrobial peptides in lipid bilayers.

To investigate the role of peptide-membrane interactions in the biological activity of cyclic cationic peptides, the conformations and interactions of four membrane-active antimicrobial peptides [based on Gramicidin S (GS)] were examined in neutral and negatively charged micelles and phospholipid vesicles, using CD and fluorescence spectroscopy and ultracentrifugation techniques. Moreover, the effects of these peptides on the release of entrapped fluorescent dye from unilamellar vesicles of phosphatidylcholine (PC) and phosphatidylethanolamine/phosphatidylglycerol (PE/PG) were studied. The cyclic peptides include GS10 [Cyclo(VKLdYP)2], GS12 [Cyclo(VKLKdYPKVKLdYP)], GS14 [Cyclo(VKLKVdYPLKVKLdYP)] and [d-Lys]4GS14 [Cyclo(VKLdKVdYPLKVKLdYP)] (underlined residues are d-amino acids), were different in their ring size, structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. Interaction of the peptides with the zwitterionic PC and negatively charged PE/PG vesicles were distinct from each other. The hydrophobic interaction seems to be the dominant factor in the hemolytic activity of the peptides, as well as their interaction with the PC vesicles. A combination of electrostatic and hydrophobic interactions of the peptides induces aggregation and fusion in PE/PG vesicles with different propensities in the order: [d-Lys]4GS14 > GS14 > GS12 > GS10. GS10 and GS14 are apparently located in the deeper levels of the membrane interfaces and closer to the hydrophobic core of the bilayers, whereas GS12 and [d-Lys]4GS14 reside closer to the outer boundary of the interface. Because of differing modes of interaction of the cyclic cationic peptides with lipid bilayers, the mechanism of their biological activity (and its relation to peptide-lipid interaction) proved to be versatile and complex, and dependent on the biophysical properties of both the peptides and membranes.     

Pore formation of phospholipid membranes by the action of two hemolytic arachnid peptides of different size

Pin2 and Oxki1 are cationic amphipathic peptides that permeate lipid membranes through formation of pores. Their mechanism of binding to phosphocholine (PC) membranes differs. Spin-probe experiments showed that both Pin2 and Oxki1 penetrate the lipid membrane of small unilamellar vesicles (SUVs). Moreover, the leakage of calcein and dextrans from PC vesicles showed that Pin2 agrees with the accumulation of peptides on lipid membranes and form pores of different size. On the other hand, Oxki1 did not act strictly cooperatively and form pores of limited size.     

Pore formation of phospholipid membranes by the action of two hemolytic arachnid peptides of different size

Pin2 and Oxki1 are cationic amphipathic peptides that permeate lipid membranes through formation of pores. Their mechanism of binding to phosphocholine (PC) membranes differs. Spin-probe experiments showed that both Pin2 and Oxki1 penetrate the lipid membrane of small unilamellar vesicles (SUVs). Moreover, the leakage of calcein and dextrans from PC vesicles showed that Pin2 agrees with the accumulation of peptides on lipid membranes and form pores of different size. On the other hand, Oxki1 did not act strictly cooperatively and form pores of limited size.     

Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R(6)GC-NH(2) and K(6)GC-NH(2), are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall barrier function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV-1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer.     

Interaction of small peptides with lipid bilayers.

Molecular dynamics simulations of the tripeptide Ala-Phe-Ala-O-tert-butyl interacting with dimyristoylphosphatidylcholine lipid bilayers have been carried out. The lipid and aqueous environments of the peptide, the alkyl chain order, and the lipid and peptide dynamics have been investigated with use of density profiles, radial distribution functions, alkyl chain order parameter profiles, and time correlation functions. It appears that the alkyl chain region accommodates the peptides in the bilayer with minimal perturbation to this region. The peptide dynamics in the bilayer bound form has been compared with that of the free peptide in water. The peptide structure does not vary on the simulation time scale (of the order of hundreds of picoseconds) compared with the solution structure in which a random structure is observed.     

Anti-Legionella activity of staphylococcal hemolytic peptides

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic.     

Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.     

pH-dependent self-association of influenza hemagglutinin fusion peptides in lipid bilayers

We have recently designed a host-guest peptide system that allows us to quantitatively measure the energetics of interaction of viral fusion peptides with lipid bilayers. Here, we show that fusion peptides of influenza hemagglutinin reversibly associate with one another at membrane surfaces above critical surface concentrations, which range from one to five peptides per 1000 lipids in the systems that we investigated. It is further demonstrated by using circular dichroism and Fourier transform infrared spectroscopy that monomeric peptides insert into the bilayers in a predominantly alpha-helical conformation, whereas self-associated fusion peptides adopt predominantly antiparallel beta-sheet structures at the membrane surface. The two forms are readily interconvertible and the equilibrium between them is determined by the pH and ionic strength of the surrounding solution. Lowering the pH favors the monomeric alpha-helical conformation, whereas increasing the ionic strength shifts the equilibrium towards the membrane-associated beta-aggregates. The binding data are interpreted in terms of a cooperative binding model that yields free energies of insertion and free energies of self-association for each of the peptides studied at pH 7.4 and pH 5. At pH 5 and 35 mM ionic strength, the insertion energy of the 20 residue influenza hemagglutinin fusion peptide is -7.2 kcal/mol and the self-association energy is -1.9 kcal/mol. We propose that self-association of fusion peptides could be a major driving force for recruiting a small number of hemagglutinin trimers into a fusion site.     

Simulation studies of the interaction of antimicrobial peptides and lipid bilayers

Experimental studies of a number of antimicrobial peptides are sufficiently detailed to allow computer simulations to make a significant contribution to understanding their mechanisms of action at an atomic level. In this review we focus on simulation studies of alamethicin, melittin, dermaseptin and related antimicrobial, membrane-active peptides. All of these peptides form amphipathic alpha-helices. Simulations allow us to explore the interactions of such peptides with lipid bilayers, and to understand the effects of such interactions on the conformational dynamics of the peptides. Mean field methods employ an empirical energy function, such as a simple hydrophobicity potential, to provide an approximation to the membrane. Mean field approaches allow us to predict the optimal orientation of a peptide helix relative to a bilayer. Molecular dynamics simulations that include an atomistic model of the bilayer and surrounding solvent provide a more detailed insight into peptide-bilayer interactions. In the case of alamethicin, all-atom simulations have allowed us to explore several steps along the route from binding to the membrane surface to formation of transbilayer ion channels. For those antimicrobial peptides such as dermaseptin which prefer to remain at the surface of a bilayer, molecular dynamics simulations allow us to explore the favourable interactions between the peptide helix sidechains and the phospholipid headgroups.     

A theoretical model for the association of amphiphilic transmembrane peptides in lipid bilayers

A theoretical model is proposed for the association of trans-bilayer peptides in lipid bilayers. The model is based on a lattice model for the pure lipid bilayer, which accounts accurately for the most important conformational states of the lipids and their mutual interactions and statistics. Within the lattice formulation the bilayer is formed by two independent monolayers, each represented by a triangular lattice, on which sites the lipid chains are arrayed. The peptides are represented by regular objects, with no internal flexibility, and with a projected area on the bilayer plane corresponding to a hexagon with seven lattice sites. In addition, it is assumed that each peptide surface at the interface with the lipid chains is partially hydrophilic, and therefore interacts with the surrounding lipid matrix via selective anisotropic forces. The peptides would therefore assemble in order to shield their hydrophilic residues from the hydrophobic surroundings. The model describes the self-association of peptides in lipid bilayers via lateral and rotational diffusion, anisotropic lipid-peptide interactions, and peptide-peptide interactions involving the peptide hydrophilic regions. The intent of this model study is to analyse the conditions under which the association of trans-bilayer and partially hydrophilic peptides (or their dispersion in the lipid matrix) is lipid-mediated, and to what extent it is induced by direct interactions between the hydrophilic regions of the peptides. The model properties are calculated by a Monte Carlo computer simulation technique within the canonical ensemble. The results from the model study indicate that direct interactions between the hydrophilic regions of the peptides are necessary to induce peptide association in the lipid bilayer in the fluid phase. Furthermore, peptides within each aggregate are oriented in such a way as to shield their hydrophilic regions from the hydrophobic environment. The average number of peptides present in the aggregates formed depends on the degree of mismatch between the peptide hydrophobic length and the lipid bilayer hydrophobic thickness: The lower the degree of mismatch is the higher this number is. Received: 30 December 1996 / Accepted: 9 May 1997     

Action of two classes of channel-forming synthetic peptides on lipid bilayers

Single channel conductances were observed in lipid bilayers by the action of three Dialkylamide derivatives of Gramicidin and two polymers of the tripeptide (Ala-Ala-Gly). The temperature and thickness dependence showed conformational changes indicating multiple states of the helical structure.     

A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides.

The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC50/IC50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC50 = 0.6 microM) and had the lowest therapeutic index (HC50/IC50 = 0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.     

Membrane activity of the pentaene macrolide didehydroroflamycoin in model lipid bilayers

Didehydroroflamycoin (DDHR), a recently isolated member of the polyene macrolide family, was shown to have antibacterial and antifungal activity. However, its mechanism of action has not been investigated. Antibiotics from this family are amphiphilic; thus, they have membrane activity, their biological action is localized in the membrane, and the membrane composition and physical properties facilitate the recognition of a particular compound by the target organism. In this work, we use model lipid membranes comprised of giant unilamellar vesicles (GUVs) for a systematic study of the action of DDHR. In parallel, experiments are conducted using filipin III and amphotericin B, other members of the family, and the behavior observed for DDHR is described in the context of that of these two heavily studied compounds. The study shows that DDHR disrupts membranes via two different mechanisms and that the involvement of these mechanisms depends on the presence of cholesterol. The leakage assays performed in GUVs and the conductance measurements using black lipid membranes (BLM) reveal that the pores that develop in the absence of cholesterol are transient and their size is dependent on the DDHR concentration. In contrast, cholesterol promotes the formation of more defined structures that are temporally stable.     

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.     

Strength of integration of transmembrane alpha-helical peptides in lipid bilayers as determined by atomic force spectroscopy

In this study we address the stability of integration of proteins in membranes. Using dynamic atomic force spectroscopy, we measured the strength of incorporation of peptides in lipid bilayers. The peptides model the transmembrane parts of alpha-helical proteins and were studied in both ordered peptide-rich and unordered peptide-poor bilayers. Using gold-coated AFM tips and thiolated peptides, we were able to observe force events which are related to the removal of single peptide molecules out of the bilayer. The data demonstrate that the peptides are very stably integrated into the bilayer and that single barriers within the investigated region of loading rates resist their removal. The distance between the ground state and the barrier for peptide removal was found to be 0.75 +/- 0.15 nm in different systems. This distance falls within the thickness of the interfacial layer of the bilayer. We conclude that the bilayer interface region plays an important role in stably anchoring transmembrane proteins into membranes.     

Clustering of tetrameric influenza M2 peptides in lipid bilayers investigated by 19F solid-state NMR

The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating, and carries out membrane scission to enable virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 catalyzes membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use ¹⁹F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 peptide and CF₃-Phe47 labeled M2 peptide and measuring F-CF₃ cross peaks in 2D ¹⁹F¹⁹F correlation spectra, we show that M2 tetramers form nanometer-scale clusters in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. ¹H¹⁹F correlation peaks between lipid chain protons and fluorinated Phe47 indicate that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static ³¹P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.     

Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes.