A solvent model for simulations of peptides in bilayers. I. Membrane-promoting alpha-helix formation

We describe an efficient solvation model for proteins. In this model atomic solvation parameters imitating the hydrocarbon core of a membrane, water, and weak polar solvent (octanol) were developed. An optimal number of solvation parameters was chosen based on analysis of atomic hydrophobicities and fitting experimental free energies of gas-cyclohexane, gas-water, and octanol-water transfer for amino acids. The solvation energy term incorporated into the ECEPP/2 potential energy function was tested in Monte Carlo simulations of a number of small peptides with known energies of bilayer-water and octanol-water transfer. The calculated properties were shown to agree reasonably well with the experimental data. Furthermore, the solvation model was used to assess membrane-promoting alpha-helix formation. To accomplish this, all-atom models of 20-residue homopolypeptides-poly-Leu, poly-Val, poly-Ile, and poly-Gly in initial random coil conformation-were subjected to nonrestrained Monte Carlo conformational search in vacuo and with the solvation terms mimicking the water and hydrophobic parts of the bilayer. All the peptides demonstrated their largest helix-forming tendencies in a nonpolar environment, where the lowest-energy conformers of poly-Leu, Val, Ile revealed 100, 95, and 80% of alpha-helical content, respectively. Energetic and conformational properties of Gly in all environments were shown to be different from those observed for residues with hydrophobic side chains. Applications of the solvation model to simulations of peptides and proteins in the presence of membrane, along with limitations of the approach, are discussed.     

Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity

The amphiphilic anionic peptides E5 and E5L can mimic the fusogenic activity of influenza hemagglutinin(HA). These peptides induced fusion of egg yolk phosphatidylcholine small or large unilamellar vesicles only at acidic pH in a similar manner to viral HA. Acetylation or acetimidylation of the N-terminus of the peptides drastically reduced the fusion activity of the intact peptides, while C-terminal amidation left the activity unchanged. The binding assay suggested that the interaction of the modified peptides with lipid membranes was almost unchanged in comparison with those of the parent peptides, and the CD spectra showed that these peptides were alpha-helical. The results showed the importance of the N-terminus of the peptides on the membrane fusion activity, although why the N-terminal modifications affect the activity is still unclear.     

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.     

Free-energy determinants of alpha-helix insertion into lipid bilayers.

A detailed treatment is provided of the various free-energy terms that contribute to the transfer of a polyalanine alpha-helix from the aqueous phase into lipid bilayers. In agreement with previous work, the hydrophobic effect is found to provide the major driving force for helix insertion. However, an opposing effect of comparable magnitude is also identified and is attributed to the large free-energy penalty associated with the desolvation of peptide hydrogen bonds on transfer to the low dielectric environment of the bilayer. Lipid perturbation effects as well as the entropy loss associated with helix immobilization in the bilayer are also evaluated. Two configurations of a membrane-bound 25mer polyalanine helix were found to be lower in free energy than the isolated helix in the aqueous phase. The first corresponds to the case of vertical insertion, in which a helix terminus protrudes from each side of the bilayer. The second minimum is for the case of horizontal insertion, for which the helix is adsorbed upon the surface of the bilayer. The calculated free-energy minima are found to be in good agreement with recent measurements of related systems. Large free-energy barriers resulting from desolvation of unsatisfied hydrogen-bonding groups at the helix termini are obtained for both insertion processes. The barriers for insertion are significantly reduced if the helix termini are assumed to be "capped" through the formation of hydrogen bonds with polar sidechains. For uncapped helices, our results support recently proposed models in which helices are inserted by first adsorbing on the membrane surface and then having one terminus "swing around" so as to penetrate 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.     

Plasticity of influenza haemagglutinin fusion peptides and their interaction with lipid bilayers

A detailed molecular dynamics study of the haemagglutinin fusion peptide (N-terminal 20 residues of the HA2 subunits) in a model bilayer has yielded useful information about the molecular interactions leading to insertion into the lipids. Simulations were performed on the native sequence, as well as a number of mutant sequences, which are either fusogenic or nonfusogenic. For the native sequence and fusogenic mutants, the N-terminal 11 residues of the fusion peptides are helical and insert with a tilt angle of approximately 30 degrees with respect to the membrane normal, in very good agreement with experimental data. The tilted insertion of the native sequence peptide leads to membrane bilayer thinning and the calculated order parameters show larger disorder of the alkyl chains. These results indicate that the lipid packing is perturbed by the fusion peptide and could be used to explain membrane fusion. For the nonfusogenic sequences investigated, it was found that most of them equilibrate parallel to the interface plane and do not adopt a tilted conformation. The presence of a charged residue at the beginning of the sequence (G1E mutant) resulted in a more difficult case, and the outcomes do not fall straightforwardly into the general picture. Sequence searches have revealed similarities of the fusion peptide of influenza haemagglutinin with peptide sequences such as segments of porin, amyloid alpha eta peptide, and a peptide from the prion sequence. These results confirm that the sequence can adopt different folds in different environments. The plasticity and the conformational dependence on the local environment could be used to better understand the function of fusion peptides.     

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.     

Rapid membrane fusion of individual virus particles with supported lipid bilayers

Many enveloped viruses employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus proteins, and buffer conditions in membrane fusion.     

Folding of apocytochrome c in lipid micelles: formation of alpha-helix precedes membrane insertion.

Apocytochrome c, which in aqueous solution is largely unstructured, acquires a highly alpha-helical structure upon interaction with lipid. The alpha-helix content induced in apocytochrome c depends on the lipid system, and this folding process is driven by both electrostatic and hydrophobic lipid-protein interactions. The folding kinetic mechanism of apocytochrome c induced by zwitterionic micelles of lysophosphatidylcholine (L-PC), predominantly driven by hydrophobic lipid-protein interactions, was investigated by fluorescence stopped-flow measurements of Trp 59 and fluorescein-phosphatidylethanolamine-(FPE) labeled micelles, in combination with stopped-flow far-UV circular dichroism. It was found that formation of the alpha-helical structure of apocytochrome c precedes membrane insertion. The unfolded state in solution (U(W)) binds to the micelle surface in a helical conformation (I(S)) and is followed by insertion into the lipid micelle, i.e., formation of the final helical state H(L). Binding of apocytochrome c to the lipid micelle (U(W) --> I(S)) is concurrent with formation of a large fraction (75-100%, depending on lipid concentration) of the alpha-helical structure of the final lipid-inserted state H(L). The highly helical intermediate I(S) is formed on the time scale of 3-12 ms, depending on lipid concentration, and inserts into the lipid micelle (I(S) --> H(L)) in the time range of approximately 200 ms to >1 s, depending on lipid-to-protein ratio. The final lipid-inserted helical state H(L) in L-PC micelles has an alpha-helix content approximately 65% of that of cytochrome c in solution and has no compact stable tertiary structure as revealed by circular dichroism results.     

Structure stability of lytic peptides during their interactions with lipid bilayers.

In this work, molecular dynamics simulations were used to examine the consequences of a variety of analogs of cecropin A on lipid bilayers. Analog sequences were constructed by replacing either the N- or C-terminal helix with the other helix in native or reverse sequence order, by making palindromic peptides based on both the N- and C-terminal helices, and by deleting the hinge region. The structure of the peptides was monitored throughout the simulation. The hinge region appeared not to assist in maintaining helical structure but help in motion flexibility. In general, the N-terminal helix of peptides was less stable than the C-terminal one during the interaction with anionic lipid bilayers. Sequences with hydrophobic helices tended to regain helical structure after an initial loss while sequences with amphipathic helices were less able to do this. The results suggests that hydrophobic design peptides have a high structural stability in an anionic membrane and are the candidates for experimental investigation.     

Voltage-dependent formation of gramicidin channels in lipid bilayers.

The formation kinetics of gramicidin A channels in lipid bilayer membranes has been characterized as a function of voltage for different solution conditions and membrane composition. The frequency of channel events was measured during the application of voltage ramps and counted in given intervals, a procedure that eliminated the effects of drift in gramicidin concentration. The formation rate was found to increase strongly with voltages up to approximately 50 mV and then to level off slightly. The shape of the voltage dependence was independent of lipid solvent and ramp speed but differed for different ions and different solution concentrations. This suggested an ion occupancy effect on the formation rate that was further supported by the fact that the minimum of the formation rate was shifted toward the equilibrium potential in asymmetric solution concentrations. The effects are explained in terms of a model that contains two contributions to the voltage dependence, a voltage-dependent ion binding to the monomers and a polarization of monomers by the applied electric field and by the occupied ions. The theory is found to give a good fit to experimental data.     

The influence of cholesterol on the interaction of HIV gp41 membrane proximal region-derived peptides with lipid bilayers

A small amino acid sequence (LWYIK) inside the HIV-1 gp41 ectodomain membrane proximal region (MPR) is commonly referred to as a cholesterol-binding domain. To further study this unique and peculiar property we have used fluorescence spectroscopy techniques to unravel the membrane interaction properties of three MPR-derived synthetic peptides: the membrane proximal region peptide-complete (MPRP-C) which corresponds to the complete MPR; the membrane proximal region peptide-short (MPRP-S), which corresponds to the last five MPR amino acid residues (the putative cholesterol-binding domain) and the membrane proximal region peptide-intermediate (MPRP-I), which corresponds to the MPRP-C peptide without the MPRP-S sequence. MPRP-C and MPRP-I membrane interaction is largely independent of the membrane phase. Membrane interaction of MPRP-S occurs for fluid phase membranes but not in gel phase membranes or cholesterol-containing bilayers. The gp41 ectodomain MPR may have a very specific function in viral fusion through the concerted and combined action of cholesterol-binding and non-cholesterol-binding domains (i.e. domains corresponding to MPRP-S and MPRP-I, respectively).     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Protein-mediated intermembrane contact facilitates fusion of lipid vesicles with planar bilayers

Fusion of phospholipid vesicles with planar bilayer membranes occurs provided there is an intermembrane contact, which can be mediated by phospholipid-binding proteins, even in the absence of calcium. The firm attachment phase is then followed by the osmotically-driven fusion. These results show that hydrophobic proteins (not necessarily Ca²⁺-binding proteins) may enhance fusion by promoting contact of membranes. Such proteins may operate synergistically with Ca²⁺ to reduce the threshold concentration of Ca²⁺ needed for fusion of biological membranes. Protein-mediated intermembrane contact resulting in fusion may play a crucial role in the regulation and catalysis of biological fusion events.     

Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers

We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles.     

Detection of transient capacitance increase associated with channel formation in lipid bilayers.

The insertion of alpha- and beta-latrotoxins and sea anemone (Radianthus macrodactilus) toxin into bilayer lipid membranes (BLMs) was investigated using the method of simultaneous conductance/capacitance measurement. All the toxins investigated induced capacitance changes which preceded toxin-induced conductance increases. The processes that may underlie the observed effect are discussed.     

The interaction of viral fusion peptides with lipid membranes

In this paper, we studied fusogenic peptides of class I-III fusion proteins, which are relevant to membrane fusion for certain enveloped viruses, in contact with model lipid membranes. We resolved the vertical structure and examined the adsorption or penetration behavior of the fusogenic peptides at phospholipid Langmuir monolayers with different initial surface pressures with x-ray reflectometry. We show that the fusion loops of tick-borne encephalitis virus (TBEV) glycoprotein E and vesicular stomatitis virus (VSV) G-protein are not able to insert deeply into model lipid membranes, as they adsorbed mainly underneath the headgroups with only limited penetration depths into the lipid films. In contrast, we observed that the hemagglutinin 2 fusion peptide (HA2-FP) and the VSV-transmembrane domain (VSV-TMD) can penetrate deeply into the membranes. However, in the case of VSV-TMD, the penetration was suppressed already at low surface pressures, whereas HA2-FP was able to insert even into highly compressed films. Membrane fusion is accompanied by drastic changes of the membrane curvature. To investigate how the peptides affect the curvature of model lipid membranes, we examined the effect of the fusogenic peptides on the equilibration of cubic monoolein structures after a phase transition from a lamellar state induced by an abrupt hydrostatic pressure reduction. We monitored this process in presence and absence of the peptides with small-angle x-ray scattering and found that HA2-FP and VSV-TMD drastically accelerate the equilibration, while the fusion loops of TBEV and VSV stabilize the swollen state of the lipid structures. In this work, we show that the class I fusion peptide of HA2 penetrates deeply into the hydrophobic region of membranes and is able to promote and accelerate the formation of negative curvature. In contrast, we found that the class II and III fusion loops of TBEV and VSV tend to counteract negative membrane curvature.     

Interaction of mutant influenza virus hemagglutinin fusion peptides with lipid bilayers: probing the role of hydrophobic residue size in the central region of the fusion peptide.

The amino-terminal region of the membrane-anchored subunit of influenza virus hemagglutinin, the fusion peptide, is crucial for membrane fusion of this virus. The peptide is extruded from the interior of the protein and inserted into the lipid bilayer of the target membrane upon induction of a conformational change in the protein by low pH. Although the effects of several mutations in this region on the fusion behavior and the biophysical properties of the corresponding peptides have been studied, the structural requirements for an active fusion peptide have still not been defined. To probe the sensitivity of the fusion peptide structure and function to small hydrophobic perturbations in the middle of the hydrophobic region, we have individually replaced the alanine residues in positions 5 and 7 with smaller (glycine) or bulkier (valine) hydrophobic residues and measured the extent of fusion mediated by these hemagglutinin constructs as well as some biophysical properties of the corresponding synthetic peptides in lipid bilayers. We find that position 5 tolerates a smaller and position 7 a larger hydrophobic side chain. All peptides contained segments of alpha-helical (33-45%) and beta-strand (13-16%) conformation as determined by CD and ATR-FTIR spectroscopy. The order parameters of the peptide helices and the lipid hydrocarbon chains were determined from measurements of the dichroism of the respective infrared absorption bands. Order parameters in the range of 0.0-0.6 were found for the helices of these peptides, which indicate that these peptides are most likely aligned with their alpha-helices at oblique angles to the membrane normal. Some (mostly fusogenic) peptides induced significant increases of the order parameter of the lipid hydrocarbon chains, suggesting that the lipid bilayer becomes more ordered in the presence of these peptides, possibly as a result of dehydration at the membrane surface.     

Antimicrobial peptides temporins B and L induce formation of tubular lipid protrusions from supported phospholipid bilayers

The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < approximately 1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to approximately 1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (>/=+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections.