Molecular theory of lipid-protein interaction and the Lalpha-HII transition.

We present a molecular-level theory for lipid-protein interaction and apply it to the study of lipid-mediated interactions between proteins and the protein-induced transition from the planar bilayer (Lalpha) to the inverse-hexagonal (HII) phase. The proteins are treated as rigid, membrane-spanning, hydrophobic inclusions of different size and shape, e.g., "cylinder-like," "barrel-like," or "vase-like." We assume strong hydrophobic coupling between the protein and its neighbor lipids. This means that, if necessary, the flexible lipid chains surrounding the protein will stretch, compress, and/or tilt to bridge the hydrophobic thickness mismatch between the protein and the unperturbed bilayer. The system free energy is expressed as an integral over local molecular contributions, the latter accounting for interheadgroup repulsion, hydrocarbon-water surface energy, and chain stretching-tilting effects. We show that the molecular interaction constants are intimately related to familiar elastic (continuum) characteristics of the membrane, such as the bending rigidity and spontaneous curvature, as well as to the less familiar tilt modulus. The equilibrium configuration of the membrane is determined by minimizing the free energy functional, subject to boundary conditions dictated by the size, shape, and spatial distribution of inclusions. A similar procedure is used to calculate the free energy and structure of peptide-free and peptide-rich hexagonal phases. Two degrees of freedom are involved in the variational minimization procedure: the local length and local tilt angle of the lipid chains. The inclusion of chain tilt is particularly important for studying noncylindrical (for instance, barrel-like) inclusions and analyzing the structure of the HII lipid phase; e.g., we find that chain tilt relaxation implies strong faceting of the lipid monolayers in the hexagonal phase. Consistent with experiment, we find that only short peptides (large negative mismatch) can induce the Lalpha --> HII transition. At the transition, a peptide-poor Lalpha phase coexists with a peptide-rich HII phase.     

Correlation between fusogenicity of synthetic modified peptides corresponding to the NH2-terminal extremity of simian immunodeficiency virus gp32 and their mode of insertion into the lipid bilayer: an infrared spectroscopy study.

The amino-terminal extremity of the simian immunodeficiency virus (SIV) transmembrane protein (gp32) has been shown to play a pivotal role in cell-virus fusion and syncytium formation. We provide here evidence of a correlation between the structure and orientation of the modified SIV fusion peptide after insertion into the lipid membrane and its fusogenic activity. The sequence of the wild-type SIV peptide has been modified in such a way that the calculated angles of insertion correspond to an oblique, parallel, or normal orientation with respect to the lipid-water interface. Fourier transform infrared spectroscopy was used to gain experimental informations about the structures and orientations, of the membrane-inserted peptides with respect to the lipid acyl chains. The peptides adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with large unilamellar liposomes, this beta sheet is partly converted into alpha helix. The ability of the modified peptides to promote lipid mixing was assessed by a fluorescence energy transfer assay. The data provide evidence that alpha-helix formation is not sufficient to induce lipid mixing and that the fusogenic activity of the peptide depends on its orientation in the lipid bilayer.     

Stalk model of membrane fusion: solution of energy crisis.

Membrane fusion proceeds via formation of intermediate nonbilayer structures. The stalk model of fusion intermediate is commonly recognized to account for the major phenomenology of the fusion process. However, in its current form, the stalk model poses a challenge. On one hand, it is able to describe qualitatively the modulation of the fusion reaction by the lipid composition of the membranes. On the other, it predicts very large values of the stalk energy, so that the related energy barrier for fusion cannot be overcome by membranes within a biologically reasonable span of time. We suggest a new structure for the fusion stalk, which resolves the energy crisis of the model. Our approach is based on a combined deformation of the stalk membrane including bending of the membrane surface and tilt of the hydrocarbon chains of lipid molecules. We demonstrate that the energy of the fusion stalk is a few times smaller than those predicted previously and the stalks are feasible in real systems. We account quantitatively for the experimental results on dependence of the fusion reaction on the lipid composition of different membrane monolayers. We analyze the dependence of the stalk energy on the distance between the fusing membranes and provide the experimentally testable predictions for the structural features of the stalk intermediates.     

Structural intermediates in influenza haemagglutinin-mediated fusion

Fusion pore formation in the haemagglutinin (HA)-mediated fusion is a culmination of a multistep process, which involves low-pH triggered refolding of HA and rearrangement of membrane lipid bilayers. This rearrangement was arrested or slowed down by either altering lipid composition of the membranes, or lowering the density of HA, and/ or temperature. The results suggest that fusion starts with the lateral assembly of activated HA into multimeric complexes surrounding future fusion sites. The next fusion stage involves hemifusion, i.e. merger of only contacting membrane monolayers. Lysophosphatidylcholine reversibly arrests fusion prior to this hemifusion stage. In the normal fusion pathway, hemifusion is transient and is not accompanied by any measurable transfer of lipid probes between the membranes. A temperature of 4degreeC stabilizes this `restricted hemifusion' intermediate. The restriction of lipid flow through the restricted hemifusion site is HA-dependent and can be released by partial cleaving of low pH-forms of HA with mild proteinase K treatment. Lipid effects indicate that fusion proceeds through two different lipid-involving intermediates, which are characterized by two opposite curvatures of the lipid monolayer. Hemifusion involves formation of a stalk, a local bent connection between the outer membrane monolayers. Fusion pore formation apparently involves bending of the inner membrane monolayers, which come together in hemifusion. To couple low pH-induced refolding of HA with lipid rearrangements, it is proposed that the extension of the alpha -helical coiled coil of HA pulls fusion peptides inserted into the HA-expressing membrane and locally bends the membrane into a saddle-like shape. Elastic energy drives self-assembly of these HA-containing membrane elements into a ring-like complex and causes the bulging of the host membrane into a dimple growing towards the target membrane. Bending stresses in the lipidic top of the dimple facilitate membrane fusion.     

Structural intermediates in influenza haemagglutinin-mediated fusion.

Fusion pore formation in the haemagglutinin (HA)-mediated fusion is a culmination of a multistep process, which involves low-pH triggered refolding of HA and rearrangement of membrane lipid bilayers. This rearrangement was arrested or slowed down by either altering lipid composition of the membranes, or lowering the density of HA, and/or temperature. The results suggest that fusion starts with the lateral assembly of activated HA into multimeric complexes surrounding future fusion sites. The next fusion stage involves hemifusion, i.e. merger of only contacting membrane monolayers. Lysophosphatidylcholine reversibly arrests fusion prior to this hemifusion stage. In the normal fusion pathway, hemifusion is transient and is not accompanied by any measurable transfer of lipid probes between the membranes. A temperature of 4 degrees C stabilizes this 'restricted hemifusion' intermediate. The restriction of lipid flow through the restricted hemifusion site is HA-dependent and can be released by partial cleaving of low pH-forms of HA with mild proteinase K treatment. Lipid effects indicate that fusion proceeds through two different lipid-involving intermediates, which are characterized by two opposite curvatures of the lipid monolayer. Hemifusion involves formation of a stalk, a local bent connection between the outer membrane monolayers. Fusion pore formation apparently involves bending of the inner membrane monolayers, which come together in hemifusion. To couple low pH-induced refolding of HA with lipid rearrangements, it is proposed that the extension of the alpha-helical coiled coil of HA pulls fusion peptides inserted into the HA-expressing membrane and locally bends the membrane into a saddle-like shape. Elastic energy drives self-assembly of these HA-containing membrane elements into a ring-like complex and causes the bulging of the host membrane into a dimple growing towards the target membrane. Bending stresses in the lipidic top of the dimple facilitate membrane fusion.     

13C and 15N NMR evidence for peripheral intercalation of uniformly labeled fusogenic peptides incorporated in a biomimetic membrane

Membrane fusion requires drastic and transient changes of bilayer curvature and here we have studied the interaction of three de novo designed synthetic hydrophobic peptides with a biomimetic three-lipid mixture by solid state NMR. An experimental approach is presented for screening of peptide-lipid interactions and their aggregation, and their embedding in a biomimetic membrane system using established proton-decoupled 13C,15N and proton spin diffusion heteronuclear 1H-13C correlation NMR methods at high magnetic field. Experiments are presented for a set of de-novo designed fusion peptides in interaction with their lipid environment. The data provide additional support for the transmembrane model for the least fusogenic peptide, L16, while the peripheral intercalation model is preferred for the fusogenic peptides LV16 and LV16G8P9. This contributes to converging evidence that peripheral intercalation is both necessary and sufficient to trigger the fusion process for a lipid mixture close to a critical point for phase separation across the bilayer.     

Comparative structural studies of Vpu peptides in phospholipid monolayers by x-ray scattering

Vpu is an 81-residue HIV-1 accessory protein, its transmembrane and cytoplasmic domains each responsible for one of its two functions. Langmuir monolayers of phospholipid incorporating a membrane protein with a unidirectional vectorial orientation, on a semiinfinite aqueous subphase, provide one "membranelike" environment for the protein. The cytoplasmic domain's interaction with the surface of the phospholipid monolayer in determining the tertiary structure of the peptide within the monolayer was investigated, employing a comparative structural study of Vpu with its submolecular fragments Tm and TmCy truncated to different extents in the cytoplasmic domain, via synchrotron x-ray scattering utilizing a new method of analysis. Localizations of the transmembrane and cytoplasmic domains within the monolayer profile structure were similar for all three proteins, the hydrophobic transmembrane helix within the hydrocarbon chain region tilted with respect to the monolayer plane and the helices of the cytoplasmic domains lying on the surface of the headgroups parallel to the monolayer plane. The thickness of the hydrocarbon chain region, determined by the tilt of the hydrocarbon chains and transmembrane domain with respect to the monolayer plane, was slightly different for Tm, TmCy, and Vpu systematically with protein/lipid mole ratio. Localization of the helices in the cytoplasmic domains of the three proteins relative to the headgroups depends on their extents and amphipathicities. Thus, the interaction of the cytoplasmic domain of Vpu on the surface may affect the tilt of the transmembrane helix within the hydrocarbon chain region in determining its tertiary structure in the membrane.     

Oblique membrane insertion of viral fusion peptide probed by neutron diffraction

Fusion peptides mimic the membrane fusion activities of the larger viral proteins from which they derive their sequences. A possible mode of activity involves their oblique insertion into lipid bilayers, causing membrane disruption by promoting highly curved hemifusion intermediates, leading to fusion. We have determined the location and orientation of the simian immunodeficiency virus (SIV) fusion peptide in planar lipid bilayers using neutron lamellar diffraction. The helical axis of the peptide adopts an angle of 55 degrees relative to the membrane normal, while it positions itself nearest the lipid bilayer surface. This is the first direct observation of the structural interaction between a fusion peptide and a phospholipid bilayer.     

Atomistic-level study of the interactions between hIAPP protofibrils and membranes: Influence of pH and lipid composition

The pathology of type 2 diabetes mellitus is associated with the aggregation of human islet amyloid polypeptide (hIAPP) and aggregation-mediated membrane disruption. The interactions of hIAPP aggregates with lipid membrane, as well as the effects of pH and lipid composition at the atomic level, remain elusive. Herein, using molecular dynamics simulations, we investigate the interactions of hIAPP protofibrillar oligomers with lipids, and the membrane perturbation that they induce, when they are partially inserted in an anionic dipalmitoyl-phosphatidylglycerol (DPPG) membrane or a mixed dipalmitoyl-phosphatidylcholine (DPPC)/DPPG (7:3) lipid bilayer under acidic/neutral pH conditions. We observed that the tilt angles and insertion depths of the hIAPP protofibril are strongly correlated with the pH and lipid composition. At neutral pH, the tilt angle and insertion depth of hIAPP protofibrils at a DPPG bilayer reach ~52° and ~1.62 nm with respect to the membrane surface, while they become ~77° and ~1.75 nm at a mixed DPPC/DPPG membrane. The calculated tilt angle of hIAPP at DPPG membrane is consistent with a recent chiral sum frequency generation spectroscopic study. The acidic pH induces a smaller tilt angle of ~40° and a shallower insertion depth (~1.24 nm) of hIAPP at the DPPG membrane surface, mainly due to protonation of His18 near the turn region. These differences mainly result from a combination of distinct electrostatic, van der Waals, hydrogen bonding and salt-bridge interactions between hIAPP and lipid bilayers. The hIAPP-membrane interaction energy analysis reveals that besides charged residues K1, R11 and H18, aromatic residues Phe15 and Phe23 also exhibit strong interactions with lipid bilayers, revealing the crucial role of aromatic residues in stabilizing the membrane-bound hIAPP protofibrils. hIAPP-membrane interactions disturb the lipid ordering and the local bilayer thickness around the peptides. Our results provide atomic-level information of membrane interaction of hIAPP protofibrils, revealing pH-dependent and membrane-modulated hIAPP aggregation at the early stage.     

Interaction of Influenza Virus Fusion Peptide with Lipid Membranes: Effect of Lysolipid

The effect of lysophosphatidylcholine (LPC) on lipid vesicle fusion and leakage induced by influenza virus fusion peptides and the peptide interaction with lipid membranes were studied by using fluorescence spectroscopy and monolayer surface tension measurements. It was confirmed that the wild-type fusion peptide-induced vesicle fusion rate increased several-fold between pH 7 and 5, unlike a mutated peptide, in which valine residues were substituted for glutamic acid residues at positions 11 and 15. This mutated peptide exhibited a much greater ability to induce lipid vesicle fusion and leakage but in a less pH-dependent manner compared to the wild-type fusion peptide. The peptide-induced vesicle fusion and leakage were well correlated with the degree of interaction of these peptides with lipid membranes, as deduced from the rotational correlation time obtained for the peptide tryptophan fluorescence. Both vesicle fusion and leakage induced by the peptides were suppressed by LPC incorporated into lipid vesicle membranes in a concentration-dependent manner. The rotational correlation time associated with the peptide’s tryptophan residue, which interacts with lipid membranes containing up to 25 mole % LPC, was virtually the same compared to lipid membranes without LPC, indicating that LPC-incorporated membrane did not affect the peptide interaction with the membrane. The adsorption of peptide onto a lipid monolayer also showed that the presence of LPC did not affect peptide adsorption.     

Small scale membrane mechanics

Large scale changes to lipid bilayer shapes are well represented by the Helfrich model. However, there are membrane processes that take place at smaller length scales that this model cannot address. In this work, we present a one-dimensional continuum model that captures the mechanics of the lipid bilayer membrane at the length scale of the lipids themselves. The model is developed using the Cosserat theory of surfaces with lipid orientation, or ‘tilt’, as the fundamental degree of freedom. The Helfrich model can be recovered as a special case when the curvatures are small and the lipid tilt is everywhere zero. We use the tilt model to study local membrane deformations in response to a protein inclusion. Parameter estimates and boundary conditions are obtained from a coarse-grained molecular model using dissipative particle dynamics (DPD) to capture the same phenomenon. The continuum model is able to reproduce the membrane bending, stretch and lipid tilt as seen in the DPD model. The lipid tilt angle relaxes to the bulk tilt angle within 5–6 nm from the protein inclusion. Importantly, for large tilt gradients induced by the proteins, the tilt energy contribution is larger than the bending energy contribution. Thus, the continuum model of tilt accurately captures behaviors at length scales shorter than the membrane thickness.     

Lipids in biological membrane fusion

The results reviewed suggest that membrane fusion in diverse biological fusion reactions involves formation of some specific intermediates: stalks and pores. Energy of these intermediates and, consequently, the rate and extent of fusion depend on the propensity of the corresponding monolayers of membranes to bend in the required directions.Proteins and peptides can control the bending energy of membrane monolayers in a number of ways. Monolayer lipid composition may be altered by different phospholipases [50, 85, 90], flipases and translocases [4, 50]. Proteins and peptides can change monolayer spontaneous curvature or hydrophobic void energy by direct interaction with membrane lipids [20, 32, 111]. Proteins may also provide some barriers for lipid diffusion in the plane of the monolayer [83, 141]. If diffusion of lipids at some specific membrane sites (e.g., in the vicinity of fusion protein) is somehow hindered, the energy of the bent fusion intermediates would reflect the elastic properties of these particular sites rather than the spontaneous curvature of the whole monolayers. Proteins may deform membranes while bringing them locally into close contact. The alteration of the geometric (external) curvature will certainly change the elastic energy of the initial state and, thus affect the energetic barriers of the formation of the intermediates [143]. In addition, the area and the energy of the stalk can be reduced by preliminary bending of the contacting membranes [111]. The possible effects of proteins and polymers on local elastic properties and local shapes of the membranes have been recently analyzed [22, 39, 45, 63]. These studies may provide a good basis for future development of theoretical models of protein-mediated fusion.     

The hydrophobic insertion mechanism of membrane curvature generation by proteins

A wide spectrum of intracellular processes is dependent on the ability of cells to dynamically regulate membrane shape. Membrane bending by proteins is necessary for the generation of intracellular transport carriers and for the maintenance of otherwise intrinsically unstable regions of high membrane curvature in cell organelles. Understanding the mechanisms by which proteins curve membranes is therefore of primary importance. Here we suggest, for the first time to our knowledge, a quantitative mechanism of lipid membrane bending by hydrophobic or amphipathic rodlike inclusions which simulate amphipathic α-helices—structures shown to sculpt membranes. Considering the lipid monolayer matrix as an anisotropic elastic material, we compute the intramembrane stresses and strains generated by the embedded inclusions, determine the resulting membrane shapes, and the accumulated elastic energy. We characterize the ability of an inclusion to bend membranes by an effective spontaneous curvature, and show that shallow rodlike inclusions are more effective in membrane shaping than are lipids having a high propensity for curvature. Our computations provide experimentally testable predictions on the protein amounts needed to generate intracellular membrane shapes for various insertion depths and membrane thicknesses. We also predict that the ability of N-BAR domains to produce membrane tubules in vivo can be ascribed solely to insertion of their amphipathic helices.     

The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.     

Molecular dynamics simulations of the anchoring and tilting of the lung-surfactant peptide SP-B1-25 in palmitic acid monolayers

We have performed molecular dynamics simulations of multiple copies of the lung-surfactant peptide SP-B1-25 in a palmitic acid (PA) monolayer. SP-B1-25 is a shorter version of lung-surfactant protein B, an important component of lung surfactant. Up to 30 ns simulations of 20 wt % SP-B1-25 in the PA monolayers were performed with different surface areas of PA, extents of PA ionization, and various initial configurations of the peptides. Starting with initial peptide orientation perpendicular to the monolayer, the predicted final tilt angles average 54 degrees approximately 62 degrees with respect to the monolayer normal, similar to those measured experimentally by Lee et al. (Biophysical Journal. 2001. Synchrotron x-ray study of lung surfactant-specific protein SP-B in lipid monolayers. 81:572-585). In their final conformations, hydrogen-bond analysis and amino acid mutation studies show that the peptides are anchored by hydrogen bond interactions between the cationic residues Arg-12 and Arg-17 and the hydrogen bond acceptors of the ionized PA headgroup, and the tilt angle is affected by the interactions of Tyr-7 and Gln-19 with the PA headgroup. Our work indicates that the factors controlling orientation of small peptides in lipid layers can now be uncovered through molecular dynamics simulations.     

Fusogenic activity of SIV (simian immunodeficiency virus) peptides located in the GP32 NH2 terminal domain

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.     

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.     

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.     

Unbinding-binding transition induced by molecular snaps in model membranes

We have used a lamellar phase made of a nonionic surfactant, dodecane and water, as a model membrane to investigate its interactions with macromolecular inclusions bringing together two membranes, i.e., acting as macromolecular snaps. In systems devoid of inclusions, the interlamellar distance depends on the total volume fraction of membranes Phi. We show that, in presence of a transmembrane protein, or of several de novo designed peptides of different length and composition, the lamellar phase undergoes a binding transition. Under such conditions, the interlamellar distance is no longer proportional to Phi(-1), but rather to the surface concentration of snaps within the membrane. It also appears that, in the presence of the hydrophobic segment of peptide snaps, the length of the inclusions must be at least equal to the hydrophobic length of the membrane to be active. Experimental results have been precisely fitted to a model of thermally stabilized membranes, decorated with snaps. However, in the presence of inclusions, the parameter describing the interactions between membranes, has to take into account the length of the inclusion to preserve good predictive capabilities.