Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.     

Conformations and orientations of a signal peptide interacting with phospholipid monolayers

The interaction of a chemically synthesized 25-residue signal peptide of LamB protein from Escherichia coli with phospholipids has been studied with a film balance technique. The conformation, orientation, and concentration of the peptides in lipid monolayers have been determined from polarized infrared spectroscopy, ultraviolet spectroscopy, and assay of 14C-labeled peptide in transferred films. When the LamB signal peptide is injected into the subphase under a phosphatidylethanolamine-phosphatidylglycerol monolayer at low initial pressure, insertion of a portion of the peptide into the lipid film is evidenced by a rapid rise in film pressure. Spectroscopic results obtained on films transferred to quartz plates and Ge crystals show that the peptide is a mixture of alpha-helix and beta-conformation where the long axis of the alpha-helix penetrates the monolayer plane and the beta-structure is coplanar with the film. By contrast, when peptide is injected under lipid at high initial pressure, no pressure rise is observed, and the spectroscopic results show the presence of only beta-structure which is coplanar with the monolayer. The spectroscopic and radioassay results are all consistent with the picture of a peptide anchored to the monolayer through electrostatic binding with a helical portion inserted into the lipid region of the monolayer and a beta-structure portion resident in the aqueous phase. The negative charges on the lipid molecules are roughly neutralized by the positive charges of the peptide.     

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers. Here, the membrane interaction of the 65-kDa activated Cry4Ba protein with the lipid monolayers, comprising dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine, and cholesterol (Chol), was studied using Langmuir-Blodgett technique. The interactions of the Cry4Ba protein with the lipid monolayers were measured from the surface pressure versus area isotherms of the protein-lipid monolayers. The increase in the mean molecular area was demonstrated as an incorporation of the protein into lipid monolayers. The insertion of the Cry4Ba protein was monitored by measuring as an increase of the surface pressure at constant molecular area. For a given monolayer, the membrane insertion of the Cry4Ba reduced as the initial surface pressure increased. The Cry4Ba protein showed a strong preference of an insertion towards a Chol monolayer. In addition, the mixed monolayers of Chol showed an enhanced effect on the insertion kinetics of Cry4Ba into lipid films, suggesting its involvement in the modulation of the protein insertion. These findings provide the first evidence that the Cry4Ba protein is capable of inserting itself into lipid monolayers, depending on the packing density of the monolayers. Our results also indicate that only a limited part of the protein is likely to be involved in the insertion.     

Oscillations in the lateral pressure of lipid monolayers induced by nonlinear chemical dynamics of the second messengers MARCKS and protein kinase C

The binding of the MARCKS peptide to the lipid monolayer containing PIP₂ increases the lateral pressure of the monolayer. The unbinding dynamics modulated by protein kinase C leads to oscillations in lateral pressure of lipid monolayers. These periodic dynamics can be attributed to changes in the crystalline lipid domain size. We have developed a mathematical model to explain these observations based on the changes in the physical structure of the monolayer by the translocation of MARCKS peptide. The model indicates that changes in lipid domain size drives these oscillations. The model is extended to an open system that sustains chemical oscillations.     

Sticholysin I–membrane interaction: An interplay between the presence of sphingomyelin and membrane fluidity

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.     

GsMTx4: Mechanism of Inhibiting Mechanosensitive Ion Channels

GsMTx4 is a spider venom peptide that inhibits cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We synthesized six analogs with single lysine-to-glutamate substitutions and tested them against Piezo1 channels in outside-out patches and independently measured lipid binding. Four analogs had ～20% lower efficacy than the wild-type (WT) peptide. The equilibrium constants calculated from the rates of inhibition and washout did not correlate with the changes in inhibition. The lipid association strength of the WT GsMTx4 and the analogs was determined by tryptophan autofluorescence quenching and isothermal calorimetry with membrane vesicles and showed no significant differences in binding energy. Tryptophan fluorescence-quenching assays showed that both WT and analog peptides bound superficially near the lipid-water interface, although analogs penetrated deeper. Peptide-lipid association, as a function of lipid surface pressure, was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure. Analogs with compromised efficacy had pressure-area isotherms with steeper slopes in this region, suggesting tighter peptide association. The pressure-dependent redistribution of peptide between “deep” and “shallow” binding modes was supported by molecular dynamics (MD) simulations of the peptide-monolayer system under different area constraints. These data suggest a model placing GsMTx4 at the membrane surface, where it is stabilized by the lysines, and occupying a small fraction of the surface area in unstressed membranes. When applied tension reduces lateral pressure in the lipids, the peptides penetrate deeper acting as “area reservoirs” leading to partial relaxation of the outer monolayer, thereby reducing the effective magnitude of stimulus acting on the MSC gate.     

Asymmetrical membranes and surface tension

The (31)P-nuclear magnetic resonance chemical shift of phosphatidic acid in a membrane is sensitive to the lipid head group packing and can report qualitatively on membrane lateral compression near the aqueous interface. We have used high-resolution (31)P-nuclear magnetic resonance to evaluate the lateral compression on each side of asymmetrical lipid vesicles. When monooleoylphosphatidylcholine was added to the external monolayer of sonicated vesicles containing dioleoylphosphatidylcholine and dioleoylphosphatidic acid, the variation of (31)P chemical shift of phosphatidic acid indicated a lateral compression in the external monolayer. Simultaneously, a slight dilation was observed in the inner monolayer. In large unilamellar vesicles on the other hand the lateral pressure increased in both monolayers after asymmetrical insertion of monooleoylphosphatidylcholine. This can be explained by assuming that when monooleoylphosphatidylcholine is added to large unilamellar vesicles, the membrane bends until the strain is the same in both monolayers. In the case of sonicated vesicles, a change of curvature is not possible, and therefore differential packing in the two layers remains. We infer that a variation of lipid asymmetry by generating a lateral strain in the membrane can be a physiological way of modulating the conformation of membrane proteins.     

Interaction of methionine–enkephalins with raft-forming lipids: monolayers and BAM experiments

Enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are opioid peptides with proven antinociceptive action in organism. They interact with opioid receptors belonging to G-protein coupled receptor superfamily. It is known that these receptors are located preferably in membrane rafts composed mainly of sphingomyelin (Sm), cholesterol (Cho), and phosphatidylcholine. In the present work, using Langmuir’s monolayer technique in combination with Wilhelmy’s method for measuring the surface pressure, the interaction of synthetic methionine–enkephalin and its amidated derivative with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sm, and Cho, as well as with their double and triple mixtures, was studied. From the pressure/area isotherms measured, the compressional moduli of the lipids and lipid–peptide monolayers were determined. Our results showed that the addition of the synthetic enkephalins to the monolayers studied led to change in the lipid monolayers characteristics, which was more evident in enkephalinamide case. In addition, using Brewster angle microscopy (BAM), the surface morphology of the lipid monolayers, before and after the injection of both enkephalins, was determined. The BAM images showed an increase in surface density of the mixed surface lipids/enkephalins films, especially with double and triple component lipid mixtures. This effect was more pronounced for the enkephalinamide as well. These observations showed that there was an interaction between the peptides and the raft-forming lipids, which was stronger for the amidated peptide, suggesting a difference in folding of both enkephalins. Our research demonstrates the potential of lipid monolayers for elegant and simple membrane models to study lipid–peptide interactions at the plane of biomembranes.     

Lipid-like behavior of signal sequence peptides at air–water interface

Several protein transport processes in the cell are mediated by signal sequence peptides located at the N-terminal side of the mature protein sequence. To date, the specific interaction and the stability of these peptides at the amphipathic interface of biological membranes and the relevance of the peptide conformation when they interact with lipids is not clear. We report the surface properties and the peptide–lipid interaction of three signal sequence peptides at the air–NaCl 145 mM interface by using the Langmuir monolayer approach. These synthetic peptides have a natural sequence with a non-periodic amphiphilicity, where hydrophobic and hydrophilic residues are located on opposed sides of the peptide primary sequence. We show that signal sequence peptides form insoluble monolayers of high stability against lateral compression. At close packing, peptide molecular area, surface potential and the high stability of the peptide monolayer are indicative that signal sequence peptides are compatible with a β-sheet conformation at the interface. Structure was confirmed with PM-IRRAS and transmission FT-IR studies. The peptides show lateral miscibility with either POPC (a liquid-expanded lipid) or DPPC (a liquid-condensed lipid) in mixed peptide–lipid monolayers. This indicates that signal sequence peptides studied are laterally miscible with phospholipids independent of the phase state of the lipid.     

SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study.

SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.     

The membrane-binding properties of a class A amphipathic peptide

The membrane-binding properties of a class A amphipathic peptide (18D) were investigated using two different immobilized model membrane systems. The first system involved the use of surface plasmon resonance (SPR) to study the binding of 18D to dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylglycerol (DMPG), which allowed peptide binding to be monitored in real time. The SPR experiments indicated stronger binding of 18D to DMPG than DMPC, which kinetic analysis revealed was due to a faster on-rate. The second model membrane system involved immobilized membrane chromatography in which the binding of 18D to either DMPC or DMPG monolayers covalently linked to silica particles was analysed by elution chromatography. Stronger binding affinity of 18D was also obtained with the negatively charged phosphatidylglycerol (PG) monolayer compared to the phosphatidylcholine (PC) monolayer, which was consistent with the SPR results. Non-linear binding behaviour of 18D to the immobilized lipid monolayers was also observed, which suggests that the peptide undergoes conformational and orientational changes upon binding to the immobilized PC and PG ligands. Significant band broadening was also observed on both monolayers, with larger bandwidths obtained on the PC surface, indicating slower binding and orientation kinetics with the zwitterionic surface. The dependence of logk' on the percentage of methanol also demonstrated a bimodal interaction whereby hydrophobic forces predominated at higher temperatures and methanol concentrations, while at lower temperatures, electrostatic and other polar forces also made a contribution to the affinity of the peptides for the lipid monolayer particularly. Overall, these results demonstrate the complementary use of these two lipid biosensors which allows the role of hydrophobic and electrostatic forces in peptide–membrane interactions to be studied and insight gained into the kinetic factors associated with these interactions.     

Measuring protein insertion areas in lipid monolayers by fluorescence correlation spectroscopy

Membrane insertion of protein domains is an important step in many membrane remodeling processes, for example, in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. To our knowledge, this is the first optical measurement of this quantity on a system in equilibrium with direct determination of the number of inserted protein and no further assumptions concerning the binding thermodynamics. Whereas macroscopic total area changes in lipid monolayers are typically measured on a Langmuir film balance, finding the number of inserted proteins without perturbing the system and quantitating any small area changes has posed a challenge. Here, we address both issues by performing two-color fluorescence correlation spectroscopy directly on the monolayer. With a fraction of the protein being fluorescently labeled, the number of inserted proteins is determined in situ without resorting to invasive techniques such as collecting the monolayer by aspiration. The second color channel is exploited to monitor a small fraction of labeled lipids to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm² was obtained for Sar1 in monolayers from a lipid mixture typically used in COPII reconstitution experiments, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to also be applied to laterally heterogeneous monolayers and bilayers.     

Fluid and condensed ApoA-I/phospholipid monolayers provide insights into ApoA-I membrane insertion

Apolipoprotein A-I (ApoA-I) is a protein implicated in the solubilization of lipids and cholesterol from cellular membranes. The study of ApoA-I in phospholipid (PL) monolayers brings relevant information about ApoA-I/PL interactions. We investigated the influence of PL charge and acyl chain organization on the interaction with ApoA-I using dipalmitoyl-phosphatidylcholine, dioleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylglycerol monolayers coupled to ellipsometric, surface pressure, atomic force microscopy and infrared (polarization modulation infrared reflection-absorption spectroscopy) measurements. We show that monolayer compressibility is the major factor controlling protein insertion into PL monolayers and show evidence of the requirement of a minimal distance between lipid headgroups for insertion to occur, Moreover, we demonstrate that ApoA-I inserts deepest at the highest compressibility of the protein monolayer and that the presence of an anionic headgroup increases the amount of protein inserted in the PL monolayer and prevents the steric constrains imposed by the spacing of the headgroup. We also defined the geometry of protein clusters into the lipid monolayer by atomic force microscopy and show evidence of the geometry dependence upon the lipid charge and the distance between headgroups. Finally, we show that ApoA-I helices have a specific orientation when associated to form clusters and that this is influenced by the character of PL charges. Taken together, our results suggest that the interaction of ApoA-I with the cellular membrane may be driven by a mechanism that resembles that of antimicrobial peptide/lipid interaction.     

Determination of the contribution of the myristoyl group and hydrophobic amino acids of recoverin on its dynamics of binding to lipid monolayers

It has been postulated that myristoylation of peripheral proteins would facilitate their binding to membranes. However, the exact involvement of this lipid modification in membrane binding is still a matter of debate. Proteins containing a Ca(2+)-myristoyl switch where the extrusion of their myristoyl group is dependent on calcium binding is best illustrated by the Ca(2+)-binding recoverin, which is present in retinal rod cells. The parameters responsible for the modulation of the membrane binding of recoverin are still largely unknown. This study was thus performed to determine the involvement of different parameters on recoverin membrane binding. We have used surface pressure measurements and PM-IRRAS spectroscopy to monitor the adsorption of myristoylated and nonmyristoylated recoverin onto phospholipid monolayers in the presence and absence of calcium. The adsorption curves have shown that the myristoyl group and hydrophobic residues of myristoylated recoverin strongly accelerate membrane binding in the presence of calcium. In the case of nonmyristoylated recoverin in the presence of calcium, hydrophobic residues alone are responsible for its much faster monolayer binding than myristoylated and nonmyristoylated recoverin in the absence of calcium. The infrared spectra revealed that myristoylated and nonmyristoylated recoverin behave very different upon adsorption onto phospholipid monolayers. Indeed, PM-IRRAS spectra indicated that the myristoyl group allows a proper orientation and organization as well as faster and stronger binding of myristoylated recoverin to lipid monolayers compared to nonmyristoylated recoverin. Simulations of the spectra have allowed us to postulate that nonmyristoylated recoverin changes conformation and becomes hydrated at large extents of adsorption as well as to estimate the orientation of myristoylated recoverin with respect to the monolayer plane. In addition, adsorption measurements and electrophoresis of trypsin-treated myristoylated recoverin in the presence of zinc or calcium demonstrated that recoverin has a different conformation but a similar extent of monolayer binding in the presence of such ions.     

Insertion of lipidated Ras proteins into lipid monolayers studied by infrared reflection absorption spectroscopy (IRRAS)

Ras proteins have to be associated with the inner leaflet of the plasma membrane to perform their signaling functions. This membrane targeting and binding is controlled by post-translational covalent attachment of farnesyl and palmitoyl chains to cysteines in the membrane anchor region of the N- and H-Ras isoforms. Two N-Ras lipoproteins were investigated, namely a farnesylated and hexadecylated protein, presenting the natural hydrophobic modifications and a doubly hexadecylated construct, respectively. The proteins are surface active and form a Gibbs monolayer at the air-D2O interface. The contours of the amide-I bands were analyzed using infrared reflection absorption spectroscopy (IRRAS). Langmuir monolayers of a mixture of POPC, brain sphingomyelin, and cholesterol were used as half of a model biomembrane to study the insertion of these N-Ras proteins. They insert with their hydrophobic anchors into lipid monolayers but at higher surface pressures (30 mN/m); the farnesylated and hexadecylated protein desorbs completely from the monolayer, whereas the doubly hexadecylated protein remains incorporated. During the insertion process, changes in the orientation of the protein secondary structure were detected by comparison with simulated IRRA spectra, based on the information on the relative orientation of the secondary structure elements from the protein crystal structure data.     

Lipid-Membrane Affinity of Chimeric Metal-binding Green Fluorescent Protein

The Green Fluorescent Protein (GFP) is a useful marker to trace the expression of cellular proteins. However, little is known about changes in protein interaction properties after fusion to GFP. In this study, we present evidence for a binding affinity of chimeric cadmium-binding green fluorescent proteins to lipid membrane. This affinity has been observed in both cellular membranes and artificial lipid monolayers and bilayers. At the cellular level, the presence of Cd-binding peptide promoted the association of the chimeric GFP onto the lipid membrane, which declined the fluorescence emission of the engineered cells. Binding affinity to lipid membranes was further investigated using artificial lipid bilayers and monolayers. Small amounts of the chimeric GFP were found to incorporate into the lipid vesicles due to the high surface pressure of bilayer lipids. At low interfacial pressure of the lipid monolayer, incorporation of the chimeric Cd-binding GFP onto the lipid monolayer was revealed. From the measured lipid isotherms, we conclude that Cd-binding GFP mediates an increase in membrane fluidity and an expansion of the surface area of the lipid film. This evidence was strongly supported by epifluorescence microscopy, showing that the chimeric Cd-binding GFP preferentially binds to fluid-phase areas and defect parts of the lipid monolayer. All these findings demonstrate the hydrophobicity of the GFP constructs is mainly influenced by the fusion partner. Thus, the example of a metal-binding unit used here shines new light on the biophysical properties of GFP constructs.This revised version was published online in June 2005 with a corrected cover date.     

Lipid headgroup discrimination by antimicrobial peptide LL-37: insight into mechanism of action

Interaction of the human antimicrobial peptide LL-37 with lipid monolayers has been investigated by a range of complementary techniques including pressure-area isotherms, insertion assay, epifluorescence microscopy, and synchrotron x-ray scattering, to analyze its mechanism of action. Lipid monolayers were formed at the air-liquid interface to mimic the surface of the bacterial cell wall and the outer leaflet of erythrocyte cell membrane by using phosphatidylglycerol (DPPG), phosphatidylcholine (DPPC), and phosphatidylethanolamine (DPPE) lipids. LL-37 is found to readily insert into DPPG monolayers, disrupting their structure and thus indicating bactericidal action. In contrast, DPPC and DPPE monolayers remained virtually unaffected by LL-37, demonstrating its nonhemolytic activity and lipid discrimination. Specular x-ray reflectivity data yielded considerable differences in layer thickness and electron-density profile after addition of the peptide to DPPG monolayers, but little change was seen after peptide injection when probing monolayers composed of DPPC and DPPE. Grazing incidence x-ray diffraction demonstrated significant peptide insertion and lateral packing order disruption of the DPPG monolayer by LL-37 insertion. Epifluorescence microscopy data support these findings.     

Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.     

Thermodynamics of fusion peptide-membrane interactions

The fusion peptides of viral membrane fusion proteins play a key role in the mechanism of viral spike glycoprotein mediated membrane fusion. These peptides insert into the lipid bilayers of cellular target membranes where they adopt mostly helical secondary structures. To better understand how membranes may be converted to high-energy intermediates during fusion, it is of interest to know how much energy, enthalpy and entropy, is provided by the insertion of fusion peptides into lipid bilayers. Here, we describe a detailed thermodynamic analysis of the binding of analogues of the influenza hemagglutinin fusion peptide of different lengths and amino acid compositions. In small unilamellar vesicles, the interaction of these peptides with lipid bilayers is driven by enthalpy (-16.5 kcal/mol) and opposed by entropy (-30 cal mol(-1) K(-1)). Most of the driving force (deltaG = -7.6 kcal/mol) comes from the enthalpy of peptide insertion deep into the lipid bilayer. Enthalpic gains and entropic losses of peptide folding in the lipid bilayer cancel to a large extent and account for only about 40% of the total binding free energy. The major folding event occurs in the N-terminal segment of the fusion peptide. The C-terminal segment mainly serves to drive the N-terminus deep into the membrane. The fusion-defective mutations G1S, which causes hemifusion, and particularly G1V, which blocks fusion, have major structural and thermodynamic consequences on the insertion of fusion peptides into lipid bilayers. The magnitudes of the enthalpies and entropies of binding of these mutant peptides are reduced, their helix contents are reduced, but their energies of self-association at the membrane surface are increased compared to the wild-type fusion peptide.     

The lipid composition of a cell membrane modulates the interaction of an antiparasitic peptide at the air-water interface

The antiparasitic property of peptides is believed to be associated with their interactions with the protozoan membrane, which calls for research on the identification of membrane sites capable of peptide binding. In this study we investigated the interaction of a lipophilic glutathioine peptide known to be effective against the African Sleeping Sickness (ASS - African Trypanosomiasis) and cell membrane models represented by Langmuir monolayers. It is shown that even small amounts of the peptide affect the monolayers of some phospholipids and other lipids, which points to a significant interaction. The latter did not depend on the electrical charge of the monolayer-forming molecules but the peptide action was particularly distinctive for cholesterol + sphingomyelin monolayers that roughly resemble rafts on a cell membrane. Using in situ polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS), we found that the orientation of the peptide is affected by the phospholipids and dioctadecyldimethylammonium bromide (DODAB), but not in monolayers comprising cholesterol + sphingomyelin. In this mixed monolayer resembling rafts, the peptide still interacts and has some induced order, probably because the peptide molecules are fitted together into a compact monolayer. Therefore, the lipid composition of the monolayer modulates the interaction with the lipophilic glutathioine peptide, and this may have important implications in understanding how the peptide acts on specific sites of the protozoan membrane.