Electric field-driven transformations of a supported model biological membrane--an electrochemical and neutron reflectivity study

A mixed bilayer of cholesterol and dimyristoylphosphatidylcholine has been formed on a gold-coated block of quartz by fusion of small unilamellar vesicles. The formation of this bilayer lipid membrane on a conductive surface allowed us to study the influence of the support's surface charge on the structure and hydration of the bilayer lipid membrane. We have employed electrochemical measurements and the specular reflection of neutrons to measure the thickness and water content in the bilayer lipid membrane as a function of the charge on the support's surface. When the surface charge density is close to zero, the lipid vesicles fuse directly on the surface to form a bilayer with a small number of defects and hence small water content. When the support's surface is negatively charged the film swells and incorporates water. When the charge density is more negative than −8 μC cm⁻², the bilayer starts to detach from the metal surface. However, it remains in a close proximity to the metal electrode, being suspended on a thin cushion of the electrolyte. The field-driven transformations of the bilayer lead to significant changes in the film thicknesses. At charge densities more negative than −20 μC cm⁻², the bilayer is ~37 Å thick and this number is comparable to the thickness determined for hydrated multilayers of dimyristoylphosphatidylcholine from x-ray diffraction experiments. The thickness of the bilayer decreases at smaller charge densities to become equal to ~26 Å at zero charge. This result indicates that the tilt of the acyl chains with respect to the bilayer normal changes from ~35° to 59° by moving from high negative charges (and potentials) to zero charge on the metal.     

CGDB: a database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

Comparison of double layer potentials in lipid monolayers and lipid bilayer membranes

Summary It is shown that the Gouy-Chapman double layer analysis adequately describes the variation of the surface potential of monolayers of acidic natural lipids over a wide range of surface charge density and salt concentration. It is also shown that the potential which initially appears when an electrolyte gradient is rapidly imposed across a bilayer membrane is due to a difference in the double layer potentials on the two sides of the membrane. This conclusion follows from the fact that the observed bilayer potentials arise much more rapidly than can be accounted for by charge migration across the membrane and from the observation that the bilayer membrane concentration potentials, when measured immediately after establishment of a gradient, are equal to the surface potential change observed when the subphase concentration of a monolayer of the same lipid is changed by an amount equal to the gradient across the bilayer. The bilayer potential and monolayer potential changes, so measured, agree in a number of different electrolyte solutions over a wide range of electrolyte concentrations and surface charge densities. Because of this agreement and the applicability of the Gouy theory to monolayers, initial bilayer potentials may be calculated if the composition of the mixture used to form the membrane is known, provided that the pK's and areas of such components are available. In the absence of this information, membrane potentials may be calculated from electrophoretic data on the membrane lipid mixture; the conditions under which the latter approach is possible have been determined. The experimental results indicate that the composition of monolyers and bilayers spread from the same lipid mixture in decane are very similar, that the composition of the two types of film closely resembles the composition of the solution used to generate them, and that bilayer membranes are close-packed. The evidence further indicates that if any hydrocarbon solvent remains in these bilayers, it must be so situated that it contributes little, if anything, to the surface area. The steady state potential in the bilayer membrane system is frequently not identical with the initial potential which supports the hypothesis that in many cases only a fraction of the electrical conductance of unmodified membranes is caused by the ions which constitute the bulk electrolyte. An expression for the relationship between diffusion and double layer potentials has been derived which shows that, in the absence of any intrinsic selectivity of the hydrocarbon region of the membrane for hydrogen, hydroxyl, or impurity, the two potentials should be identical.     

Lipid bilayer disruption by oligomeric α-synuclein depends on bilayer charge and accessibility of the hydrophobic core

Soluble oligomeric aggregates of α-synuclein have been implicated to play a central role in the pathogenesis of Parkinson's disease. Disruption and permeabilization of lipid bilayers by α-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer–membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged α-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer–membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer–membrane interactions.     

Fatty acyl chain-dependent but charge-independent association of the SH4 domain of Lck with lipid membranes

The SH4 domain of Src family of nonreceptor protein tyrosine kinases represents the extreme N-terminal 1–16 amino acid region which mediates membrane association of these proteins and facilitates their functions. The SH4 domains among Src members lack well-defined sequence consensus and vary in the net charge. However, they readily anchor to the cytoplasmic face of the plasma membrane upon fatty acid acylation. Here, we report the membrane association of differentially acylated SH4 domain of Lck kinase, which has net negative charge at physiological pH. Our results suggest that despite the net negative charge, the SH4 domain of Lck associates with membranes upon fatty acid acylation. While myristoylation at the N-terminus is sufficient for providing membrane anchorage, multiple acylation determines orientation of the peptide chain with respect to the lipid bilayer. Hence, fatty acylation serves more than just a lipid anchor. It has an important role in regulating the spatial orientation of the peptide domain with respect to the lipid bilayer, which could be important for the interaction of the other domains of these kinases with their partners.     

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.     

Ion flux across lipid bilayer membranes with charged surfaces

Summary In this paper we derive expressions for the ion flux across lipid bilayer membranes with charged surfaces treating the membrane as a continuous phase interposed between two electrolyte solutions and calculating the ion flux with the Nernst-Planck equations. The theoretical results are compared with experiments of Seufert and Hashimoto on lipid bilayer membranes with charged surface active agents added to the membranes. If the charge of both membrane surfaces has the same sign the flux of the gegenions is greatly increased whereas the flux of the coions decreases to a small amount. For oppositely charged membrane surfaces the membrane behaves like a np semiconductor and typical rectification voltage-current characteristics are obtained.     

On the nanoscale relaxation dynamics of a lipid bilayer

The Seifert–Langer theory of the relaxation dynamics of a one-component bilayer lipid membrane was extended to include a term that involves the gradient of the area per lipid molecule in the bilayer free energy per molecule. The extended theory is applicable over length scales comparable with the membrane thickness.     

Effect of membrane tension on the physical properties of DOPC lipid bilayer membrane

Molecular dynamics simulations of a dioleoylphosphocholine (DOPC) lipid bilayer were performed to explore its mechanosensitivity. Variations in the bilayer properties, such as area per lipid, volume, thickness, hydration depth (HD), hydration thickness (HT), lateral diffusion coefficient, and changes in lipid structural order were computed in the membrane tension range 0 to 15dyn/cm. We determined that an increase in membrane tension results in a decrease in the bilayer thickness and HD of ~5% and ~5.7% respectively, whereas area per lipid, volume, and HT/HD increased by 6.8%, 2.4%, and 5% respectively. The changes in lipid conformation and orientation were characterized using orientational (S(2)) and deuterium (S(CD)) order parameters. Upon increase of membrane tension both order parameters indicated an increase in lipid disorder by 10-20%, mostly in the tail end region of the hydrophobic chains. The effect of membrane tension on lipid lateral diffusion in the DOPC bilayer was analyzed on three different time scales corresponding to inertial motion, anomalous diffusion and normal diffusion. The results showed that lateral diffusion of lipid molecules is anomalous in nature due to the non-exponential distribution of waiting times. The anomalous and normal diffusion coefficients increased by 20% and 52% when the membrane tension changed from 0 to 15dyn/cm, respectively. In conclusion, our studies showed that membrane tension causes relatively significant changes in the area per lipid, volume, polarity, membrane thickness, and fluidity of the membrane suggesting multiple mechanisms by which mechanical perturbation of the membrane could trigger mechanosensitive response in cells.     

Phosphorylation and dephosphorylation of membrane proteins as a possible mechanism for structural rearrangement of membrane components.

A correlation was found between dephosphorylation of chicken erythrocyte membrane proteins, aggregation of intramembrane particles, increase in the lipid bilayer phase of the membrane and exposure of membrane phospholipids toward phospholipase A and trinitrobenzene sulfonic acid. Most of the covalently bound phosphate of the membrane proteins turns over and is associated with 5 major bands. It is suggested that phosphorylation and dephosphorylation of these proteins causes changes in their charge and conformation. Such changes might affect the interaction of these proteins with the neighbouring lipids or lipoprotein complexes and results in the aggregation of intramembrane particles and relative increase in the exposed free lipid bilayer phase of the membrane.     

Membrane thickness and acyl chain length

It appears reasonable to expect that the primary result of a change in the length of the acyl chains within a lipid bilayer is a similar change in the bilayer thickness. In the present communication we draw attention to the somewhat more complicated effects which are found experimentally for phosphatidylcholine bilayers as the hydrocarbon chain is varied from twelve to eighteen carbons in length. The major change in dimension which occurs with variation in acyl chain length is the area occupied per molecule rather than the bilayer thickness. The same effect is seen with solute hydrocarbon such as hexane which partition into the membrane and cause only a small variation in membrane thickness but a large increase in the molecular area of the lipid. The origin of this effect arises from the almost isotropic distribution of the additional hydrocarbon to the lipid core of the membrane.     

How protein transmembrane segments sense the lipid environment

Integral membrane proteins have central roles in a vast number of vital cellular processes. A structural feature that most membrane proteins have in common is the presence of one or more alpha-helices with which they traverse the lipid bilayer. Because of the interaction with the surrounding lipids, the organization of these transmembrane helices will be sensitive to lipid properties like lateral packing, hydrophobic thickness, and headgroup charge. The helices may adapt to the lipids in different ways, which in turn can influence the structure and function of the intact membrane protein. In this review, we will focus on how the lipid environment influences two specific properties of transmembrane segments: their lateral association and their tilt with respect to the bilayer normal.     

Effect of membrane tension on the electric field and dipole potential of lipid bilayer membrane

The dipole potential of lipid bilayer membrane controls the difference in permeability of the membrane to oppositely charged ions. We have combined molecular dynamics (MD) simulations and experimental studies to determine changes in electric field and electrostatic potential of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer in response to applied membrane tension. MD simulations based on CHARMM36 force field showed that electrostatic potential of DOPC bilayer decreases by ~45mV in the physiologically relevant range of membrane tension values (0 to 15dyn/cm). The electrostatic field exhibits a peak (~0.8×10(9)V/m) near the water/lipid interface which shifts by 0.9? towards the bilayer center at 15dyn/cm. Maximum membrane tension of 15dyn/cm caused 6.4% increase in area per lipid, 4.7% decrease in bilayer thickness and 1.4% increase in the volume of the bilayer. Dipole-potential sensitive fluorescent probes were used to detect membrane tension induced changes in DOPC vesicles exposed to osmotic stress. Experiments confirmed that dipole potential of DOPC bilayer decreases at higher membrane tensions. These results are suggestive of a potentially new mechanosensing mechanism by which mechanically induced structural changes in the lipid bilayer membrane could modulate the function of membrane proteins by altering electrostatic interactions and energetics of protein conformational states.     

Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.     

Stable cupola-shaped bilayer lipid membranes with mobile Plateau-Gibbs border: expansion-shrinkage of membrane due to thermal transitions.

Stable bilayer lipid membranes (BLM) with mobile Plateau-Gibbs border (PGB) have been formed. The precondition of the formation was the presence of a lipid coverage on the teflon surface near the hole, where the membrane has been formed. This allowed the movement of the PGB along the teflon surface after transformation of the planar bilayer into a cupola-shaped by bowing of the bilayer due to excess hydrostatic pressure. As a result the giant bilayers were obtained with an area up to two orders larger in magnitude compared with the initial area. Changes in lipid bilayer area depend on the temperature at the phase transition of the lipid. Cooling of the expanded bilayer was followed by a significant shrinkage of the bilayer at temperatures below the main phase transition.     

Elastic deformation and failure of lipid bilayer membranes containing cholesterol.

Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the available thermal energy, implying that many molecules (approximately 50-100) may be involved in forming the defect structure that leads to failure. The area expansion modulus of extracted RBC lipids with native cholesterol was compared with recent measurements of intact RBC membrane compressibility. The natural membrane was also modeled as a simple composite made up to a compressible lipid/cholesterol matrix containing relatively incompressible transmembrane proteins. It appears that the interaction of incompressible proteins with surrounding lipid confers enhanced compressibility on the composite structure.     

Soluble amyloid oligomers increase bilayer conductance by altering dielectric structure

The amyloid hypothesis of Alzheimer's toxicity has undergone a resurgence with increasing evidence that it is not amyloid fibrils but a smaller oligomeric species that produces the deleterious results. In this paper we address the mechanism of this toxicity. Only oligomers increase the conductance of lipid bilayers and patch-clamped mammalian cells, producing almost identical current-voltage curves in both preparations. Oligomers increase the conductance of the bare bilayer, the cation conductance induced by nonactin, and the anion conductance induced by tetraphenyl borate. Negative charge reduces the sensitivity of the membrane to amyloid, but cholesterol has little effect. In contrast, the area compressibility of the lipid has a very large effect. Membranes with a large area compressibility modulus are almost insensitive to amyloid oligomers, but membranes formed from soft, highly compressible lipids are highly susceptible to amyloid oligomer-induced conductance changes. Furthermore, membranes formed using the solvent decane (instead of squalane) are completely insensitive to the presence of oligomers. One simple explanation for these effects on bilayer conductance is that amyloid oligomers increase the area per molecule of the membrane-forming lipids, thus thinning the membrane, lowering the dielectric barrier, and increasing the conductance of any mechanism sensitive to the dielectric barrier.     

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 A resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 A resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.     

Effects of cholesterol concentration on the interaction of cytarabine with lipid membranes: a molecular dynamics simulation study

Liposomal cytarabine, DepoCyt, is a chemotherapy agent which is used in cancer treatment. This form of cytarabine has more efficacy and fewer side effects relative to the other forms. Since DepoCyt contains the cytarabine encapsulated within phosphatidylcholine and the sterol molecules, we modeled dioleoylphosphatidylcholine (DOPC)/cholesterol bilayer membrane as a carrier for cytarabine to study drug–bilayer interactions. For this purpose, we performed a series of united-atom molecular dynamics (MD) simulations for 25?ns to investigate the interactions between cytarabine and cholesterol-containing DOPC lipid bilayers. Only the uncharged form of cytarabine molecule was investigated. In this study, different levels of the cholesterol content (0, 20, and 40%) were used. MD simulations allowed us to determine dynamical and structural properties of the bilayer membrane and to estimate the preferred location and orientation of the cytarabine molecule inside the bilayer membrane. Properties such as membrane thickness, area per lipid, diffusion coefficient, mass density, bilayer packing, order parameters, and intermolecular interactions were examined. The results show that by increasing the cholesterol concentration in the lipid bilayers, the bilayer thickness increases and area per lipid decreases. Moreover, in accordance with the experiments, our calculations show that cholesterol molecules have ordering effect on the hydrocarbon acyl chains. Furthermore, the cytarabine molecule preferentially occupies the polar region of the lipid head groups to form specific interactions (hydrogen bonds). Our results fully support the experimental data. Our finding about drug–bilayer interaction is crucial for the liposomal drug design.