Simulation studies of protein-induced bilayer deformations, and lipid-induced protein tilting, on a mesoscopic model for lipid bilayers with embedded proteins

Biological membranes are complex and highly cooperative structures. To relate biomembrane structure to their biological function it is often necessary to consider simpler systems. Lipid bilayers composed of one or two lipid species, and with embedded proteins, provide a model system for biological membranes. Here we present a mesoscopic model for lipid bilayers with embedded proteins, which we have studied with the help of the dissipative particle dynamics simulation technique. Because hydrophobic matching is believed to be one of the main physical mechanisms regulating lipid-protein interactions in membranes, we considered proteins of different hydrophobic length (as well as different sizes). We studied the cooperative behavior of the lipid-protein system at mesoscopic time- and lengthscales. In particular, we correlated in a systematic way the protein-induced bilayer perturbation, and the lipid-induced protein tilt, with the hydrophobic mismatch (positive and negative) between the protein hydrophobic length and the pure lipid bilayer hydrophobic thickness. The protein-induced bilayer perturbation was quantified in terms of a coherence length, xi(P), of the lipid bilayer hydrophobic thickness profile around the protein. The dependence on temperature of xi(P), and the protein tilt-angle, were studied above the main-transition temperature of the pure system, i.e., in the fluid phase. We found that xi(P) depends on mismatch, i.e., the higher the mismatch is, the longer xi(P) becomes, at least for positive values of mismatch; a dependence on the protein size appears as well. In the case of large model proteins experiencing extreme mismatch conditions, in the region next to the so-called lipid annulus, there appears an undershooting (or overshooting) region where the bilayer hydrophobic thickness is locally lower (or higher) than in the unperturbed bilayer, depending on whether the protein hydrophobic length is longer (or shorter) than the pure lipid bilayer hydrophobic thickness. Proteins may tilt when embedded in a too-thin bilayer. Our simulation data suggest that, when the embedded protein has a small size, the main mechanism to compensate for a large hydrophobic mismatch is the tilt, whereas large proteins react to negative mismatch by causing an increase of the hydrophobic thickness of the nearby bilayer. Furthermore, for the case of small, peptidelike proteins, we found the same type of functional dependence of the protein tilt-angle on mismatch, as was recently detected by fluorescence spectroscopy measurements.     

The determinants of hydrophobic mismatch response for transmembrane helices

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane α-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4 μs. We have created 25 different tryptophan-alanine-leucine transmembrane α-helical peptide systems, each composed of a hydrophobic alanine–leucine stretch, flanked by 1–4 tryptophan side chains, as well as the β-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~ 45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes.     

Rhodopsin/Lipid Hydrophobic Matching—Rhodopsin Oligomerization and Function

Lipid composition of the membrane and rhodopsin packing density strongly modulate the early steps of the visual response of photoreceptor membranes. In this study, lipid-order and bovine rhodopsin function in proteoliposomes composed of the sn-1 chain perdeuterated lipids 14:0_d27-14:1-PC, 16:0_d31-16:1-PC, 18:0_d35-18:1-PC, or 20:0_d39-20:1-PC at rhodopsin/lipid molar ratios from 1:70 to 1:1000 (mol/mol) were investigated. Clear evidence for matching of hydrophobic regions on rhodopsin transmembrane helices and hydrophobic thickness of lipid bilayers was observed from ²H nuclear magnetic resonance order parameter measurements at low rhodopsin concentrations. Thin bilayers stretched to match the length of transmembrane helices observed as increase of sn-1 chain order, while thicker bilayers were compressed near the protein. A quantitative analysis of lipid-order parameter changes suggested that the protein adjusts its conformation to bilayer hydrophobic thickness as well, which confirmed our earlier circular-dichroism measurements. Changes in lipid order parameters upon rhodopsin incorporation vanished for bilayers with a hydrophobic thickness of 27 ± 1 Å, suggesting that this is the bilayer thickness at which rhodopsin packs in bilayers at the lowest membrane perturbation. The lipid-order parameter studies also indicated that a hydrophobic mismatch between rhodopsin and lipids triggers rhodopsin oligomerization with increasing rhodopsin concentrations. Both hydrophobic mismatch and rhodopsin oligomerization result in substantial shifts of the equilibrium between the photointermediates metarhodopsin I and metarhodopsin II; increasing bilayer thickness favors formation of metarhodopsin II while oligomerization favors metarhodopsin I. The results highlight the importance of hydrophobic matching for rhodopsin structure, oligomerization, and function.     

Revisiting Hydrophobic Mismatch with Free Energy Simulation Studies of Transmembrane Helix Tilt and Rotation

Protein-lipid interaction and bilayer regulation of membrane protein functions are largely controlled by the hydrophobic match between the transmembrane (TM) domain of membrane proteins and the surrounding lipid bilayer. To systematically characterize responses of a TM helix and lipid adaptations to a hydrophobic mismatch, we have performed a total of 5.8-μs umbrella sampling simulations and calculated the potentials of mean force (PMFs) as a function of TM helix tilt angle under various mismatch conditions. Single-pass TM peptides called WALPn (n = 16, 19, 23, and 27) were used in two lipid bilayers with different hydrophobic thicknesses to consider hydrophobic mismatch caused by either the TM length or the bilayer thickness. In addition, different flanking residues, such as alanine, lysine, and arginine, instead of tryptophan in WALP23 were used to examine their influence. The PMFs, their decomposition, and trajectory analysis demonstrate that 1), tilting of a single-pass TM helix is the major response to a hydrophobic mismatch; 2), TM helix tilting up to ∼10° is inherent due to the intrinsic entropic contribution arising from helix precession around the membrane normal even under a negative mismatch; 3), the favorable helix-lipid interaction provides additional driving forces for TM helix tilting under a positive mismatch; 4), the minimum-PMF tilt angle is generally located where there is the hydrophobic match and little lipid perturbation; 5), TM helix rotation is dependent on the specific helix-lipid interaction; and 6), anchoring residues at the hydrophilic/hydrophobic interface can be an important determinant of TM helix orientation.     

Peptide Nanopores and Lipid Bilayers: Interactions by Coarse-Grained Molecular-Dynamics Simulations

A set of 49 protein nanopore-lipid bilayer systems was explored by means of coarse-grained molecular-dynamics simulations to study the interactions between nanopores and the lipid bilayers in which they are embedded. The seven nanopore species investigated represent the two main structural classes of membrane proteins (α-helical and β-barrel), and the seven different bilayer systems range in thickness from ∼28 to ∼43 Å. The study focuses on the local effects of hydrophobic mismatch between the nanopore and the lipid bilayer. The effects of nanopore insertion on lipid bilayer thickness, the dependence between hydrophobic thickness and the observed nanopore tilt angle, and the local distribution of lipid types around a nanopore in mixed-lipid bilayers are all analyzed. Different behavior for nanopores of similar hydrophobic length but different geometry is observed. The local lipid bilayer perturbation caused by the inserted nanopores suggests possible mechanisms for both lipid bilayer-induced protein sorting and protein-induced lipid sorting. A correlation between smaller lipid bilayer thickness (larger hydrophobic mismatch) and larger nanopore tilt angle is observed and, in the case of larger hydrophobic mismatches, the simulated tilt angle distribution seems to broaden. Furthermore, both nanopore size and key residue types (e.g., tryptophan) seem to influence the level of protein tilt, emphasizing the reciprocal nature of nanopore-lipid bilayer interactions.     

Orientation and dynamics of transmembrane peptides: the power of simple models

In this review we discuss recent insights obtained from well-characterized model systems into the factors that determine the orientation and tilt angles of transmembrane peptides in lipid bilayers. We will compare tilt angles of synthetic peptides with those of natural peptides and proteins, and we will discuss how tilt can be modulated by hydrophobic mismatch between the thickness of the bilayer and the length of the membrane spanning part of the peptide or protein. In particular, we will focus on results obtained on tryptophan-flanked model peptides (WALP peptides) as a case study to illustrate possible consequences of hydrophobic mismatch in molecular detail and to highlight the importance of peptide dynamics for the experimental determination of tilt angles. We will conclude with discussing some future prospects and challenges concerning the use of simple peptide/lipid model systems as a tool to understand membrane structure and function.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane alpha-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 A, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane alpha-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Lipid-protein interactions in biological membranes: a structural perspective

Lipid molecules bound to membrane proteins are resolved in some high-resolution structures of membrane proteins. An analysis of these structures provides a framework within which to analyse the nature of lipid-protein interactions within membranes. Membrane proteins are surrounded by a shell or annulus of lipid molecules, equivalent to the solvent layer surrounding a water-soluble protein. The lipid bilayer extends right up to the membrane protein, with a uniform thickness around the protein. The surface of a membrane protein contains many shallow grooves and protrusions to which the fatty acyl chains of the surrounding lipids conform to provide tight packing into the membrane. An individual lipid molecule will remain in the annular shell around a protein for only a short period of time. Binding to the annular shell shows relatively little structural specificity. As well as the annular lipid, there is evidence for other lipid molecules bound between the transmembrane α-helices of the protein; these lipids are referred to as non-annular lipids. The average thickness of the hydrophobic domain of a membrane protein is about 29 Å, with a few proteins having significantly smaller or greater thicknesses than the average. Hydrophobic mismatch between a membrane protein and the surrounding lipid bilayer generally leads to only small changes in membrane thickness. Possible adaptations in the protein to minimise mismatch include tilting of the helices and rotation of side chains at the ends of the helices. Packing of transmembrane α-helices is dependent on the chain length of the surrounding phospholipids. The function of membrane proteins is dependent on the thickness of the surrounding lipid bilayer, sometimes on the presence of specific, usually anionic, phospholipids, and sometimes on the phase of the phospholipid.     

Importance of hydrophobic matching for spontaneous insertion of a single-spanning membrane protein

In this study, we have investigated the effect of hydrophobic mismatch between the thickness of the membrane and a transmembrane segment of a protein that directly inserts into the membrane bilayer. For this purpose we used mutants of the single-spanning Pf3 coat protein that can spontaneously insert into Escherichia coli membrane vesicles and large unilamellar vesicles (LUVs). The thickness of the liposomal bilayer could be altered by using lipids with different acyl chain lengths or by incorporation of cholesterol. The insertion efficiency of the protein clearly depended on the bilayer thickness, with most efficient insertion under hydrophobic matching conditions. To discriminate between effects of length and hydrophobicity, mutants with different synthetic transmembrane segments were constructed. These mutants inserted into LUVs in a mismatch-dependent manner. However, in particular for longer and less hydrophobic mutants, most efficient insertion was generally observed in thinner bilayers than expected on the basis of hydrophobic matching.     

Sensitivity of single membrane-spanning alpha-helical peptides to hydrophobic mismatch with a lipid bilayer: effects on backbone structure, orientation, and extent of membrane incorporation

The extent of matching of membrane hydrophobic thickness with the hydrophobic length of transmembrane protein segments potentially constitutes a major director of membrane organization. Therefore, the extent of mismatch that can be compensated, and the types of membrane rearrangements that result, can provide valuable insight into membrane functionality. In the present study, a large family of synthetic peptides and lipids is used to investigate a range of mismatch situations. Peptide conformation, orientation, and extent of incorporation are assessed by infrared spectroscopy, tryptophan fluorescence, circular dichroism, and sucrose gradient centrifugation. It is shown that peptide backbone structure is not significantly affected by mismatch, even when the extent of mismatch is large. Instead, this study demonstrates that for tryptophan-flanked peptides the dominant response of a membrane to large mismatch is that the extent of incorporation is reduced, when the peptide is both too short and too long. With increasing mismatch, a smaller fraction of peptide is incorporated into the lipid bilayer, and a larger fraction is present in extramembranous aggregates. Relatively long peptides that remain incorporated in the bilayer have a small tilt angle with respect to the membrane normal. The observed effects depend on the nature of the flanking residues: long tryptophan-flanked peptides do not associate well with thin bilayers, while equisized lysine-flanked peptides associate completely, thus supporting the notion that tryptophan and lysine interact differently with membrane-water interfaces. The different properties that aromatic and charged flanking residues impart on transmembrane protein segments are discussed in relation to protein incorporation in biological systems.     

Energetics of inclusion-induced bilayer deformations.

The material properties of lipid bilayers can affect membrane protein function whenever conformational changes in the membrane-spanning proteins perturb the structure of the surrounding bilayer. This coupling between the protein and the bilayer arises from hydrophobic interactions between the protein and the bilayer. We analyze the free energy cost associated with a hydrophobic mismatch, i.e., a difference between the length of the protein's hydrophobic exterior surface and the average thickness of the bilayer's hydrophobic core, using a (liquid-crystal) elastic model of bilayer deformations. The free energy of the deformation is described as the sum of three contributions: compression-expansion, splay-distortion, and surface tension. When evaluating the interdependence among the energy components, one modulus renormalizes the other: e.g., a change in the compression-expansion modulus affects not only the compression-expansion energy but also the splay-distortion energy. The surface tension contribution always is negligible in thin solvent-free bilayers. When evaluating the energy per unit distance (away from the inclusion), the splay-distortion component dominates close to the bilayer/inclusion boundary, whereas the compression-expansion component is more prominent further away from the boundary. Despite this complexity, the bilayer deformation energy in many cases can be described by a linear spring formalism. The results show that, for a protein embedded in a membrane with an initial hydrophobic mismatch of only 1 A, an increase in hydrophobic mismatch to 1.3 A can increase the Boltzmann factor (the equilibrium distribution for protein conformation) 10-fold due to the elastic properties of the bilayer.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Influence of hydrophobic mismatch on structures and dynamics of gramicidin a and lipid bilayers

Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4–5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations—except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch model, which has implications for understanding the energetics of membrane protein-lipid interactions.     

Spring constants for channel-induced lipid bilayer deformations. Estimates using gramicidin channels.

Hydrophobic interactions between a bilayer and its embedded membrane proteins couple protein conformational changes to changes in the packing of the surrounding lipids. The energetic cost of a protein conformational change therefore includes a contribution from the associated bilayer deformation energy (DeltaGdef0), which provides a mechanism for how membrane protein function depends on the bilayer material properties. Theoretical studies based on an elastic liquid-crystal model of the bilayer deformation show that DeltaGdef0 should be quantifiable by a phenomenological linear spring model, in which the bilayer mechanical characteristics are lumped into a single spring constant. The spring constant scales with the protein radius, meaning that one can use suitable reporter proteins for in situ measurements of the spring constant and thereby evaluate quantitatively the DeltaGdef0 associated with protein conformational changes. Gramicidin channels can be used as such reporter proteins because the channels form by the transmembrane assembly of two nonconducting monomers. The monomerleft arrow over right arrow dimer reaction thus constitutes a well characterized conformational transition, and it should be possible to determine the phenomenological spring constant describing the channel-induced bilayer deformation by examining how DeltaGdef0 varies as a function of a mismatch between the hydrophobic channel length and the unperturbed bilayer thickness. We show this is possible by analyzing experimental studies on the relation between bilayer thickness and gramicidin channel duration. The spring constant in nominally hydrocarbon-free bilayers agrees well with estimates based on a continuum analysis of inclusion-induced bilayer deformations using independently measured material constants.     

Phospholipid vesicle fusion on micropatterned polymeric bilayer substrates

As an approach to create versatile model systems of the biological membrane we have recently developed a novel micropatterning strategy of substrate-supported planar lipid bilayers (SPBs) based on photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine. The micropatterned SPBs are composed of a polymeric bilayer matrix and embedded fluid lipid bilayers. In this study, we investigated the incorporation of fluid bilayers into micropatterned polymeric bilayer matrices through the adsorption and reorganization of phospholipid vesicles (vesicle fusion). Total internal reflection fluorescence microscopy observation showed that vesicle fusion started at the boundary of polymeric bilayers and propagated into the central part of lipid-free regions. On the other hand, quartz crystal microbalance with dissipation monitoring revealed that the transformation from adsorbed vesicles into SPBs was significantly accelerated for substrates with micropatterned polymeric bilayers. These results indicate that the edges of polymeric bilayers catalyze the formation of SPBs by destabilizing adsorbed vesicles and also support the premise that polymeric bilayers and embedded fluid bilayers are forming a continuous hybrid bilayer membrane, sealing energetically unfavorable bilayer edges.     

The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence

In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.     

Polymer-cushioned bilayers. I. A structural study of various preparation methods using neutron reflectometry.

This neutron reflectometry study evaluates the structures resulting from different methods of preparing polymer-cushioned lipid bilayers. Four different techniques to deposit a dimyristoylphosphatidylcholine (DMPC) bilayer onto a polyethylenimine (PEI)-coated quartz substrate were examined: 1) vesicle adsorption onto a previously dried polymer layer; 2) vesicle adsorption onto a bare substrate, followed by polymer adsorption; and 3, 4) Langmuir-Blodgett vertical deposition of a lipid monolayer spread over a polymer-containing subphase to form a polymer-supported lipid monolayer, followed by formation of the outer lipid monolayer by either 3) horizontal deposition of the lipid monolayer or 4) vesicle adsorption. We show that the initial conditions of the polymer layer are a critical factor for the successful formation of our desired structure, i.e., a continuous bilayer atop a hydrated PEI layer. Our desired structure was found for all methods investigated except the horizontal deposition. The interaction forces between these polymer-supported bilayers are investigated in a separate paper (Wong, J. Y., C. K. Park, M. Seitz, and J. Israelachvili. 1999. Biophys. J. 77:1458-1468), which indicate that the presence of the polymer cushion significantly alters the interaction potential. These polymer-supported bilayers could serve as model systems for the study of transmembrane proteins under conditions more closely mimicking real cellular membrane environments.     

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

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

Interaction of influenza virus proteins with planar bilayer lipid membranes. I. Characterization of their adsorption and incorporation into lipid bilayers

Alterations in the surface potential difference (delta U) of asolectin planar bilayer lipid membranes were measured following the adsorption of isolated matrix protein (M-protein) or neuraminidase of influenza virus. The method used was based upon measurement of the bilayer lipid membrane capacitance current second harmonic. The delta U dependence on the M-protein and neuraminidase concentration indicates different mechanisms of adsorption of these viral proteins by the lipid bilayer. The conductance (G0) dependence of the bilayer lipid membrane with different compositions on the concentration of isolated surface glycoproteins, hemagglutinin and neuraminidase, M-protein or neuraminidase was investigated. The change in G0 for M-protein was observed only after adsorption saturation had been achieved. Neuraminidase alone does not affect the membrane conductivity. The surface charge and lipid composition of the lipid bilayer influences the adsorption and incorporation of influenza virus M-protein and surface glycoproteins. The reversibility of protein incorporation into the bilayers was investigated by a perfusion technique. The results show reversibility of surface glycoprotein incorporation while M-protein binding appears to be irreversible.