Physics and biophysics of solvent induced forces: hydrophobic interactions and context-dependent hydration

Solvent induced forces (SIFs) among solutes derive from solvent structural modification due to solutes, and consequent thermodynamic drive towards minimization of related free energy costs. The role of SIFs in biomolecular conformation and function is appreciated by observing that typical SIF values fall within the 20–200 pN interval, and that proteins are stable by only a few kcal mol^–1 (1 kcal mol^–1 corresponds to 70 pN Å). Here we study SIFs, in systems of increasing complexity, using Molecular Dynamics (MD) simulations which give time- and space-resolved details on the biologically significant scale of single protein residues and sidechains. Of particular biological relevance among our results are a strong modulability of hydrophobic SIFs by electric charges and the dependence of this modulability upon charge sign. More generally, the present results extend our understanding of the recently reported strong context-dependence of SIFs and the related potential of mean force (PMF). This context-dependence can be strong enough to propagate (by relay action) along a composite solute, and to reverse SIFs acting on a given element, relative to expectations based on its specific character (hydrophobic/ philic, charged). High specificity such as that of SIFs highlighted by the present results is of course central to biological function. Biological implications of the present results cover issues such as biomolecular functional interactions and folding (including chaperoning and pathological conformational changes), coagulation, molecular recognition, effects of phosphorylation and more.     

Electrostatic interactions between the syntaxin membrane anchor and neurotransmitter passing through the fusion pore

Recent experiments have shown that flux through the fusion pore is sensitive to manipulations of the side-chain size of certain residues in the syntaxin (syx) membrane anchor. These residues were proposed to line the wall of the fusion pore of Ca(2+)-triggered exocytosis. Here we continued this line of experimentation to examine possible electrostatic interactions between the pore lining residues and the neurotransmitter norepinephrine (NE). Replacing syx pore-lining residues with aspartate enhanced NE flux above that expected for the size of the aspartate side chain. In contrast, substitution with arginine reduced NE flux below that expected for the size of its side chain. Substituting aspartate and arginine into the nonpore-lining residues did not alter the fusion pore flux. Other amino acids with ionizable side chains had variable effects. These results indicate an electrostatic interaction between the pore-lining residues of syx and NE, and provide additional evidence that the syx membrane anchor is a structural component of the fusion pore.     

Collective properties of hydration: long range and specificity of hydrophobic interactions.

We report results of molecular dynamics (MD) simulations of composite model solutes in explicit molecular water solvent, eliciting novel aspects of the recently demonstrated, strong many-body character of hydration. Our solutes consist of identical apolar (hydrophobic) elements in fixed configurations. Results show that the many-body character of PMF is sufficiently strong to cause 1) a remarkable extension of the range of hydrophobic interactions between pairs of solute elements, up to distances large enough to rule out pairwise interactions of any type, and 2) a SIF that drives one of the hydrophobic solute elements toward the solvent rather than away from it. These findings complement recent data concerning SIFs on a protein at single-residue resolution and on model systems. They illustrate new important consequences of the collective character of hydration and of PMF and reveal new aspects of hydrophobic interactions and, in general, of SIFs. Their relevance to protein recognition, conformation, function, and folding and to the observed slight yet significant nonadditivity of functional effects of distant point mutations in proteins is discussed. These results point out the functional role of the configurational and dynamical states (and related statistical weights) corresponding to the complex configurational energy landscape of the two interacting systems: biomolecule + water.     

Aqueous solubility and membrane interactions of hydrophobic peptides with peptoid tags

Lysine tagging of hydrophobic peptides of parent sequence KKAAALAAAAALAAWAALAAAKKKK-NH(2) has been shown to facilitate their synthesis and purification through water solubilization, yet not impact on the intrinsic properties of the hydrophobic core sequence with respect to its insertion into membranes in an alpha-helical conformation. However, due to their positively charged character, such peptides often become bound to phospholipid head groups in membrane surfaces, which inhibits their transbilayer insertion and/or prevents their transport across cellular bilayers. We sought to develop more neutral peptides of membrane-permeable character by replacing most Lys residues with uncharged peptoid [N-(R)glycyl] residues, which might similarly confer water solubility while retaining membrane-interactive properties of the hydrophobic core. Several "peptoid-tagged" derivatives of the parent peptide were prepared with varying peptoid content, with five of the six Lys residues replaced with peptoids Nala and/or Nval. Conformations of these peptides measured by circular dichroism spectroscopy demonstrated that these water-soluble peptides retain the alpha-helix structure in micelles (lysophosphatidylcholine and sodium dodecyl sulfate) notwithstanding the known helix-breaking capacity of the peptoid tags. Blue shifts in Trp fluorescence spectra and quenching experiments with acrylamide confirmed that peptoid-tagged peptides insert spontaneously into micellar membranes. Results suggest that upon introduction of uncharged tags, the interaction between the membrane and the peptides is dominated by the hydrophobicity of the peptide core rather than the electrostatic interactions between the Lys and the head groups of the lipids. The overall findings indicate that peptoid residues are effective surrogates for Lys as uncharged water-solubilizing tags and, as such, provide a potentially valuable feature of design of membrane-interactive peptides.     

Surface aggregation and membrane penetration by peptides: relation to pore formation and fusion

The peptide GALA undergoes a conformational change to an amphipathic alpha -helix when the pH is reduced, inducing leakage of contents from vesicles. Leakage from neutral or negativelycharged vesicles at pH 5.0 was similar and could be adequately explained by a mathematical model which assumed that GALA becomes incorporated into the vesicle bilayer and irreversibly aggregates to form a pore consisting of M =10+/-2 peptides. Increasing cholesterol content in the membranes resulted in reduced leakage, and increased reversibility of surface aggregation of the peptide. Employing fluorescently labelled peptides confirmed that the degree of reversibility of surface aggregation of GALA was significantly larger in cholesterol containing liposomes. Orientation of the peptide GALA in bilayers was determined by a bodipy-avidin/ biotin binding assay. The peptide was labelled by biotin at the N- or Cterminus and bodipy-avidin molecules were added externally or were preencapsulated in the vesicles. The peptides are arranged in the pore perpendicularly to the membrane, such that 3/4 of the N-termini are on the internal side of the membrane. The pores are stable and persist for at least 10 min. When the peptides form an aggregate of size smaller than M, the orientation of the peptide is mostly parallel to the surface and the biotinylated peptide does not translocate. When a critical size of the aggregate is attained, a rearrangement of the peptide occurs, which amounts to rapid penetration and formation of a pore structure. Induction of fusion by peptides may be antagonistic to pore formation, the outcome being dependent on vesicle aggregation.     

Protein chemistry at membrane interfaces: non-additivity of electrostatic and hydrophobic interactions

Non-specific binding of proteins and peptides to charged membrane interfaces depends upon the combined contributions of hydrophobic (DeltaG(HPhi)) and electrostatic (DeltaG(ES)) free energies. If these are simply additive, then the observed free energy of binding (DeltaG(obs)) will be given by DeltaG(obs)=DeltaG(HPhi)+DeltaG(ES), where DeltaG(HPhi)=-sigma(NP)A(NP) and DeltaG(ES)=zFphi. In these expressions, A(NP) is the non-polar accessible area, sigma(NP) the non-polar solvation parameter, z the formal peptide valence, F the Faraday constant, and phi the membrane surface potential. But several lines of evidence suggest that hydrophobic and electrostatic binding free energies of proteins at membrane interfaces, such as those associated with cell signaling, are not simply additive. In order to explore this issue systematically, we have determined the interfacial partitioning free energies of variants of indolicidin, a cationic proline-rich antimicrobial peptide. The synthesized variants of the 13 residue peptide covered a wide range of hydrophobic free energies, which allowed us to examine the effect of hydrophobicity on electrostatic binding to membranes formed from mixtures of neutral and anionic lipids. Although DeltaG(obs) was always a linear function of DeltaG(HPhi), the slope depended upon anionic lipid content: the slope was 1.0 for pure, zwitterionic phosphocholine bilayers and 0.3 for pure phosphoglycerol membranes. DeltaG(obs) also varied linearly with surface potential, but the slope was smaller than the expected value, zF. As observed by others, this suggests an effective peptide valence z(eff) that is smaller than the formal valence z. Because of our systematic approach, we were able to establish a useful rule-of-thumb: z(eff) is reduced relative to z by about 20 % for each 3 kcal mol(-1) (1 kcal=4.184 kJ) favorable increase in DeltaG(HPhi). For neutral phosphocholine interfaces, we found that DeltaG(obs) could be predicted with remarkable accuracy using the Wimley-White experiment-based interfacial hydrophobicity scale.     

Voltage-dependent hydration and conduction properties of the hydrophobic pore of the mechanosensitive channel of small conductance

A detailed picture of water and ion properties in small pores is important for understanding the behavior of biological ion channels. Several recent modeling studies have shown that small, hydrophobic pores exclude water and ions even if they are physically large enough to accommodate them, a mechanism called hydrophobic gating. This mechanism has been implicated in the gating of several channels, including the mechanosensitive channel of small conductance (MscS). Although the pore in the crystal structure of MscS is wide and was initially hypothesized to be open, it is lined by hydrophobic residues and may represent a nonconducting state. Molecular dynamics simulations were performed on MscS to determine whether or not the structure can conduct ions. Unlike previous simulations of hydrophobic nanopores, electric fields were applied to this system to model the transmembrane potential, which proved to be important. Although simulations without a potential resulted in a dehydrated, occluded pore, the application of a potential increased the hydration of the pore and resulted in current flow through the channel. The calculated channel conductance was in good agreement with experiment. Therefore, it is likely that the MscS crystal structure is closer to a conducting than a nonconducting state.     

Spermine as a modulator of membrane fusion: interactions with acidic phospholipids

The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.     

Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion

In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca²⁺ is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca²⁺ also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca²⁺-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca²⁺-dependent SNARE binding or in Ca²⁺-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca²⁺-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca²⁺-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca²⁺-dependent membrane insertion and SNARE binding to drive fusion pore expansion.     

Forces and stresses acting on fusion pore membrane during secretion

To assess the forces and stresses present in fusion pore during secretion the stationary convective flux of lipid through a fusion pore connecting two planar membranes under different tensions was investigated through computer simulations. The physics of the problem is described by Navier-Stokes equations, and the convective flux of lipid was evaluated using finite element method. Each of the membrane monolayer is considered separately as an isotropic, homogeneous and incompressible viscous medium with the same viscosity. The difference in membrane tensions, which is simulated as the pressure difference at two ends of each monolayer, is the driving force of the lipid flow. The two monolayers interact by sliding past each other with inter-monolayer frictional viscosity. Fluid velocity, pressure, shear and normal stresses, viscous and frictional dissipations and forces were calculated to evaluate where the fusion pore will deform, extend (or compress) and dilate. The pressure changes little in the planar sections, whereas in the toroidal section the change is rapid. The magnitude of lipid velocity peaks at the pore neck. The radial lipid velocity is zero at the neck, has two peaks one on each side of the pore neck, and diminishes without going to zero in planar parts of two monolayers. The peaks are of opposite signs due to the change of direction of lipid flow. The axial velocity is confined to the toroidal section, peaks at the neck and is clearly greater in the outer monolayer. As a result of the spatially highly uneven lipid flow the membrane is under a significant stress, shear and normal. The shear stress, which indicates where the membrane will deform without changing the volume, has two peaks placed symmetrically about the neck. The normal stress shows where the membrane may extend or compress. Both, the radial and axial normal stresses are negative (extensive) in the upper toroidal section and positive (compressive) in the lower toroidal section. The pressure difference determines lipid velocity and velocity dependent variables (shear as well as normal axial and radial stresses), but also contributes directly to the force on the membranes and critically influences where and to what extent the membrane will deform, extend or dilate. The viscosity coefficient (due to friction of one element of lipid against neighboring ones), and frictional coefficient (due to friction between two monolayers sliding past each other) further modulate some variables. Lipid velocity rises as pressure difference increases, diminishes as the viscosity coefficient rises but is unaffected by the frictional coefficient. The shear and normal stresses rise as pressure difference increases, but the change of the viscosity coefficients has no effect. Both the viscous dissipation (which has two peaks placed symmetrically about the neck) and much smaller frictional dissipation (which peaks at the pore neck) rise with pressure and diminish if the viscosity coefficient rises, but only the frictional dissipation increases if the frictional coefficient increases. Finally, the radial force causing pore dilatation, and which is significant only in the planar section of the vesicular membrane, is governed almost entirely by the pressure, whereas the viscosity and frictional coefficients have only a marginal effect. Many variables are altered during pore dilatation. The lipid velocity and dissipations (viscous and frictional) rise approximately linearly with pore radius, whereas the lipid mass flow increases supra-linearly owing to the combined effects of the changes in pore radius and greater lipid velocity. Interestingly the radial force on the vesicular membrane increases only marginally.     

Model membrane platforms to study protein-membrane interactions

Abstract Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

Model membrane platforms to study protein-membrane interactions

Abstract

Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

Minimal aggregate size and minimal fusion unit for the first fusion pore of influenza hemagglutinin-mediated membrane fusion

The data of Melikyan et al. (J. Gen. Physiol. 106:783, 1995) for the time required for the first measurable step of fusion, the formation of the first flickering conductivity pore between influenza hemagglutinin (HA) expressing cells and planar bilayers, has been analyzed using a new mass action kinetic model. The analysis incorporates a rigorous distinction between the minimum number of HA trimers aggregated at the nascent fusion site (which is denoted the minimal aggregate size) and the number of those trimers that must to undergo a slow essential conformational change before the first fusion pore could form (which is denoted the minimal fusion unit). At least eight (and likely more) HA trimers aggregated at the nascent fusion site. Remarkably, of these eight (or more) HAs, only two or three must undergo the essential conformational change slowly before the first fusion pore can form. Whether the conformational change of these first two or three HAs are sufficient for the first fusion pore to form or whether the remaining HAs within the aggregate must rapidly transform in a cooperative manner cannot be determined kinetically. Remarkably, the fitted halftime for the essential HA conformational change is roughly 10(4) s, which is two orders of magnitude slower than the observed halftime for fusion. This is because the HAs refold with distributed kinetics and because the conductance assay monitored the very first aggregate to succeed in forming a first fusion pore from an ensemble of hundreds or thousands (depending upon the cell line) of fusogenic HA aggregates within the area of apposition between the cell and the planar bilayer. Furthermore, the average rate constant for this essential conformational change was at least 10(7) times slower than expected for a simple coiled coil conformational change, suggesting that there is either a high free energy barrier to fusion and/or very many nonfusogenic conformations in the refolding landscape. Current models for HA-mediated fusion are examined in light of these new constraints on the early structure and evolution of the nascent fusion site. None completely comply with the data.     

Mechanism of fusion of Sendai virus: role of hydrophobic interactions and mobility constraints of viral membrane proteins. Effects of polyethylene glycol

The mechanism of Sendai virus fusion was investigated by studying the effect of the dehydrating agent polyethylene glycol (PEG) on the interaction of the virus with erythrocyte membranes. The initial rate of virus fusion, monitored continuously by a fluorescence membrane fusion assay, increases approximately 5-fold in the presence of small amounts (4%, w/v) of PEG. The polymer did not trigger a massive nonspecific fusion event, as the limited number of virus particles that fuse per erythrocyte ghost remains unaltered. A mass action kinetic analysis reveals that the binding rate constant increases approximately 1.5-fold; however, the fusion rate constant is enhanced by about an order of magnitude. The results demonstrate that hydrophobic interaction forces dominate the actual fusion step of the virus. Below about 22 degrees C, the viral membrane proteins appear to be clustered, as revealed by temperature-dependent fluorescence measurements of fluorescently tagged viral proteins. Clustering is not modulated by the presence of PEG, and fusion at those conditions is not observed. It is concluded that in addition to hydrophobic interactions, constraints in the mobility of the viral membrane proteins codetermine the fusogenic capacity of the virus. Such constraints have to be relieved in order to allow the occurrence of the hydrophobic interactions. PEG primarily affects the surface properties of the viral membrane, including the properties of the membrane glycoproteins. We hypothesize that during virus-target membrane interaction but prior to the actual fusion reaction, the fusion protein may undergo a conformational change, triggered by an enhancement in hydrophobic environment, which accounts for the need to establish close, i.e. fusion-susceptible intermembrane contact between virus and target membrane.     

The membrane dipole potential in a total membrane potential model. Applications to hydrophobic ion interactions with membranes.

The total potential energy profile for hydrophobic ion interactions with lipid bilayers can be written as the sum of four terms: the electrical Born, image and dipole contributions, and a neutral energy term. We introduce a specific model for the membrane dipole potential, treating it as a two-dimensional array of point dipoles located near each membrane-water interface. Together with specific theoretical models for the other energy terms, a total potential profile is developed that successfully describes the complete set of thermodynamic parameters for binding and translocation for the two hydrophobic ion structural analogues, tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-). A reasonable fit to the data is possible if the dipole potential energy has a magnitude of 5.5 + 0.5 kcal/mol (240 + 20 mV), positive inside, and if the neutral energy contribution for TPP+ and TPB- is -7.0 + 1.0 kcal/mol. These results may also have important implications for small ion interactions with membranes and the energetics of charged groups in membrane proteins.     

Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.     

Membrane perturbation and fusion pore formation in influenza hemagglutinin-mediated membrane fusion. A new model for fusion

Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves conformational changes in the protein that lead to the insertion of a "fusion peptide" domain of this protein into the target membrane and is thought to perturb the membrane, triggering fusion. By using whole virus, purified HA, or HA ectodomains, we found that shortly after insertion, pores of less than 26 A in diameter were formed in liposomal membranes. As measured by a novel assay, these pores stay open, or continue to close and open, for minutes to hours and persist after pH neutralization. With virus and purified HA, larger pores, allowing the leakage of dextrans, were seen at times well after insertion. For virus, dextran leakage was simultaneous with lipid mixing and the formation of "fusion pores," allowing the transfer of dextrans from the liposomal to the viral interior or vice versa. Pores did not form in the viral membrane in the absence of a target membrane. Based on these data, we propose a new model for fusion, in which HA initially forms a proteinaceous pore in the target, but not in the viral membrane, before a lipidic hemifusion intermediate is formed.     

Exocytotic fusion pore stability and topological defects in the membrane with orientational degree of ordering

Regulated exocytosis is a process that strongly depends on the formation and stability of the fusion pore. It was indicated experimentally and theoretically that narrow and highly curved fusion pore may be stabilized by accumulation of anisotropic membrane components possessing orientational ordering. On the other hand, narrow fusion pore may also undergo repetitive opening and closing, disruption in the so called kiss and run process or become completely opened in the process of full fusion of the vesicle with the membrane. In this paper we attempt to elucidate the subtle interplay between the stabilizing and destabilizing processes in the fusion neck. A possible physical mechanism which may lead to disruption of the stable fusion pore or complete fusion of the vesicle with the membrane is discussed. It is indicated that topologically driven defects of the in-plane orientational membrane ordering in the region of the fusion pore may disrupt the fusion. Alternatively, it may facilitate repetitive opening and closing of the fusion pore or induce full fusion of the vesicle with the target membrane.     

A role of complexin-lipid interactions in membrane fusion

Complexins (Cpxs) and synaptotagmins regulate calcium-dependent exocytosis. A central helix in Cpx confers specific binding to the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) fusion machinery. An accessory helix in the amino-terminal region inhibits membrane fusion by blocking SNAREpin zippering. We now show that an amphipathic helix in the carboxy-terminal region of CpxI binds lipid bilayers and affects SNARE-mediated lipid mixing in a liposome fusion assay. The substitution of a hydrophobic amino acid within the helix by a charged residue abolishes the lipid interaction and the stimulatory effect of CpxI in liposome fusion. In contrast, the introduction of the bulky hydrophobic amino acid tryptophan stimulates lipid binding and liposome fusion. This data shows that local Cpx-lipid interactions can play a role in membrane fusion.     

Interactions of peptides with liposomes: pore formation and fusion

Leakage from liposomes induced by several peptides is reviewed and a pore model is described. According to this model peptide molecules become incorporated into the vesicle bilayer and aggregate reversibly or irreversibly within the surface. When a peptide aggregate reaches a critical size, peptide translocation can occur and a pore is formed. With the peptide GALA the pores are stable and persist for at least 10 minutes. The model predicts that for a given lipid/peptide ratio, the extent of leakage should decrease as the vesicle diameter decreases, and for a given amount of peptide bound per vesicle less leakage would be observed at higher temperatures due to the increase in reversibility of surface aggregates of the peptide. Effect of membrane composition on pore formation is reviewed. When cholesterol was included in the liposomes the efficiency of inducation of leakage by the peptide GALA was reduced due to reduced binding and increased reversibility of surface aggregation of the peptide. Phospholipids which contain less ordered acyl-chains and have a slightly wedge-like shape, can better accommodate peptide surface aggregates, and consequently insertion and translocation of the peptide may be less favored. Demonstrations of antagonism between pore formation and fusion are presented. The choice of factors which promote vesicle aggregation, e.g., larger peptides, increased vesicle and peptide concentration results in enhanced vesicle fusion at the expense of formation of intravesicular pores. FTIR studies with HIV-1 fusion peptides indicate that in systems where extensive vesicle fusion occurred the beta conformation of the peptides was predominant, whereas the alpha conformation was exhibited in cases where leakage was the main outcome. Antagonism between leakage and fusion was exhibited by 1-palmitoyl-2-oleoylphosphatidylglycerol vesicles, where the order of addition of peptide (HIV(arg)) or Ca(2+)dictated whether pore formation or vesicle fusion would occur. The current study emphasizes that the addition of Ca(2+), which promotes vesicle aggregation can also reduce peptide translocation in isolated vesicles.