The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers

We used micropipette aspiration to directly measure the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2-oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols at alcohol concentrations ranging from 0.1 to 9.8 M. The order of effectiveness in decreasing mechanical properties and increasing area per molecule was butanol>propanol>ethanol>methanol, although the lysis strain was invariant to alcohol chain-length. Quantitatively, the trend in area compressibility modulus follows Traube's rule of interfacial tension reduction, i.e., for each additional alcohol CH(2) group, the concentration required to reach the same area compressibility modulus was reduced roughly by a factor of 3. We convert our area compressibility data into interfacial tension values to: confirm that Traube's rule is followed for bilayers; show that alcohols decrease the interfacial tension of bilayer-water interfaces less effectively than oil-water interfaces; determine the partition coefficients and standard Gibbs adsorption energy per CH(2) group for adsorption of alcohol into the lipid headgroup region; and predict the increase in area per headgroup as well as the critical radius and line tension of a membrane pore for each concentration and chain-length of alcohol. The area expansion predictions were confirmed by direct measurements of the area expansion of vesicles exposed to flowing alcohol solutions. These measurements were fitted to a membrane kinetic model to find membrane permeability coefficients of short-chain alcohols. Taken together, the evidence presented here supports a view that alcohol partitioning into the bilayer headgroup region, with enhanced partitioning as the chain-length of the alcohol increases, results in chain-length-dependent interfacial tension reduction with concomitant chain-length-dependent reduction in mechanical moduli and membrane thickness.     

Interaction of salt with ether- and ester-linked phospholipid bilayers

A distinguishing feature of Archaeal plasma membranes is that their phospholipids contain ether-links, as opposed to bacterial and eukaryotic plasma membranes where phospholipids primarily contain ester-links. Experiments show that this chemical difference in headgroup-tail linkage does produce distinct differences in model bilayer properties. Here we examine the effects of salt on bilayer structure in the case of an ether-linked lipid bilayer. We use molecular dynamics simulations and compare equilibrium properties of two model lipid bilayers in NaCl salt solution – POPC and its ether-linked analog that we refer to as HOPC. We make the following key observations. The headgroup region of HOPC “adsorbs” fewer ions compared to the headgroup region of POPC. Consistent with this, we note that the Debye screening length in the HOPC system is ∼ 10% shorter than that in the POPC system. Herein, we introduce a protocol to identify the lipid-water interfacial boundary that reproduces the bulk salt distribution consistent with Gouy-Chapman theory. We also note that the HOPC bilayer has excess solvent in the headgroup region when compared to POPC, coinciding with a trough in the electrostatic potential. Waters in this region have longer autocorrelation times and smaller lateral diffusion rates compared to the corresponding region in the POPC bilayer, suggesting that the waters in HOPC are more strongly coordinated to the lipid headgroups. Furthermore, we note that it is this region of tightly coordinated waters in the HOPC system that has a lower density of Na⁺ ions. Based on these observations we conclude that an ether-linked lipid bilayer has a lower binding affinity for Na⁺ compared to an ester-linked lipid bilayer.     

Structural determinants of water permeability through the lipid membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.     

Size distribution of barrel-stave aggregates of membrane peptides: influence of the bilayer lateral pressure profile

Some membrane peptides, such as Alamethicin, form barrel-stave aggregates with a broad probability distribution of size (number of peptides in the aggregate). This distribution has been shown to depend on the characteristics of the lipid bilayer. A mechanism for this influence is suggested, in analogy to earlier work on the effects of changes in bilayer composition on conformational equilibria in membrane proteins, that is based on coupling of shifts in the distribution of lateral pressures in the bilayer to depth-dependent changes in the lateral excluded area that accompanies the formation of an aggregate. Thermodynamic analysis is coupled with a simple geometric model of aggregates of kinked cylindrical peptides and with results of previously calculated lateral pressure distributions to predict the effects of changes in bilayer characteristics on aggregate size distributions, in qualitative agreement with experimental results.     

Effects of alcohols on fluorescence anisotropies of diphenylhexatriene and its derivatives in bovine blood platelets: relationships of the depth-dependent change in membrane fluidity by alcohols with their effects on platelet aggregation and adenylate cyclase activity.

The effects of three short-chain alkyl alcohols and benzyl alcohol on the membrane fluidity of bovine blood platelets were investigated by studies on the fluorescence anisotropies of diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and its anionic propionic acid derivative (DPH-PA). These alcohols decreased the fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane, in concentration ranges that inhibited platelet aggregation. On the other hand, they had little or no effects on the fluorescence anisotropy of DPH-PA which is thought to be located in the interfacial region of the lipid bilayer. Likewise, they had little or no effects on the fluorescence anisotropy of TMA-DPH, which is also thought to be located in the interfacial region of the lipid bilayer, either when the probe was located in the outer layer of the plasma membrane or when the probe was located in the inner membrane compartment. These results suggest that alcohols mainly increase the fluidity in the central region of the lipid bilayer. Consistent with their effects on the fluorescence anisotropy of DPH, these alcohols increased the intracellular cyclic AMP concentration. Thus alcohols may inhibit platelet function due to stimulation of adenylate cyclase, which is mediated by perturbation of the central region of the membrane lipid bilayer.     

Relationship of alcohol-induced changes in Mg2+-ATPase activity of rabbit intestinal brush border membrane with changes in fluidity of its lipid bilayer

We examined the effects of seven n-alkyl alcohols (from n-butyl to n-undecyl alcohol), isoamyl alcohol and benzyl alcohol on the activity of membrane enzyme Mg²⁺-ATPase of the rabbit small intestinal brush border membrane. Their relationships with the changes in the fluidity of the membrane lipid bilayer were examined through studies on the fluorescence anisotropies of diphenylhexatriene (DPH) and its ionic derivatives. Good linear correlations were found both between the partition coefficients of the alcohols and their concentrations causing similar decreases in the activity of Mg²⁺-ATPase and between their partition coefficients and the alcohol-induced changes in fluorescence anisotropies. Within the concentration range of the alcohols tested, including isoamyl alcohol and benzyl alcohol, the decreases in activity of the membrane enzyme Mg²⁺-ATPase clearly corresponded with the decreases in fluorescence anisotropy of DPH, which is thought to be located within the hydrophobic core of the membrane. From these findings, one possible explanation is that inhibition of this enzyme by the alcohols is due to perturbation of the lipid bilayer of the brush border membrane.The authors thank M. Takano, PhD and Y. Tomita, PhD, Department of Pharmacy, University Hospital of Kyoto University, for instruction in preparation of the brush border membrane vesicles. This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture (05671795 and 06304044) and Takeda Science Foundation.     

Bilayer undulation dynamics in unilamellar phospholipid vesicles: Effect of temperature,cholesterol and trehalose

We report a combined dynamic light scattering (DLS) and neutron spin-echo (NSE) study on the local bilayer undulation dynamics of phospholipid vesicles composed of 1,2-dimyristoyl-glycero-3-phosphatidylcholine (DMPC) under the influence of temperature and the additives cholesterol and trehalose. The additives affect vesicle size and self-diffusion. Mechanical properties of the membrane and corresponding bilayer undulations are tuned by changing lipid headgroup or acyl chain properties through temperature or composition. On the local length scale, changes at the lipid headgroup influence the bilayer bending rigidity κ less than changes at the lipid acyl chain: We observe a bilayer softening around the main phase transition temperature T_m of the single lipid system, and stiffening when more cholesterol is added, in concordance with literature. Surprisingly, no effect on the mechanical properties of the vesicles is observed upon the addition of trehalose.     

How lipids affect the activities of integral membrane proteins

The activities of integral membrane proteins are often affected by the structures of the lipid molecules that surround them in the membrane. One important parameter is the hydrophobic thickness of the lipid bilayer, defined by the lengths of the lipid fatty acyl chains. Membrane proteins are not rigid entities, and deform to ensure good hydrophobic matching to the surrounding lipid bilayer. The structure of the lipid headgroup region is likely to be important in defining the structures of those parts of a membrane protein that are located in the lipid headgroup region. A number of examples are given where the conformation of the headgroup-embedded region of a membrane protein changes during the reaction cycle of the protein; activities of such proteins might be expected to be particularly sensitive to lipid headgroup structure. Differences in hydrogen bonding potential and hydration between the headgroups of phosphatidycholines and phosphatidylethanolamines could be important factors in determining the effects of these lipids on protein activities, as well as any effects related to the tendency of the phosphatidylethanolamines to form a curved, hexagonal H(II) phase. Effects of lipid structure on protein aggregation and helix-helix interactions are also discussed, as well as the effects of charged lipids on ion concentrations close to the surface of the bilayer. Interpretations of lipid effects in terms of changes in protein volume, lipid free volume, and curvature frustration are also described. Finally, the role of non-annular, or 'co-factor' lipids, tightly bound to membrane proteins, is described.     

Role of phosphatidylglycerols in the stability of bacterial membranes

An extensive 100-ns molecular dynamics simulation of lipid bilayer composed of mixture of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) was performed to elucidate the role of PGs to the stability of bacterial membranes. In addition, a control simulation of pure PE over 150 ns was performed. We observed that PGs decrease both the PE headgroup protrusions into the water phase, and the PE headgroup motion along bilayer normal. The above effects are caused by stronger inter-lipid interactions in the mixed bilayer: the number of hydrogen bonds created by PEs is 34% higher in the mixed than in the pure bilayer. Another contribution is due to the numerous ion-mediated inter-lipid links, which strongly enhance interface stability. That provides a plausible mechanism for preventing lipid desorption from the membrane, for example, under the influence of an organic solvent. A more compact and less dynamic interface structure also decreases membrane permeability. That provides a possible mechanism for stabilizing, e.g., bacterial membranes.     

Alcohol's effects on lipid bilayer properties

Alcohols are known modulators of lipid bilayer properties. Their biological effects have long been attributed to their bilayer-modifying effects, but alcohols can also alter protein function through direct protein interactions. This raises the question: Do alcohol''s biological actions result predominantly from direct protein-alcohol interactions or from general changes in the membrane properties? The efficacy of alcohols of various chain lengths tends to exhibit a so-called cutoff effect (i.e., increasing potency with increased chain length, which that eventually levels off). The cutoff varies depending on the assay, and numerous mechanisms have been proposed such as: limited size of the alcohol-protein interaction site, limited alcohol solubility, and a chain-length-dependent lipid bilayer-alcohol interaction. To address these issues, we determined the bilayer-modifying potency of 27 aliphatic alcohols using a gramicidin-based fluorescence assay. All of the alcohols tested (with chain lengths of 1–16 carbons) alter the bilayer properties, as sensed by a bilayer-spanning channel. The bilayer-modifying potency of the short-chain alcohols scales linearly with their bilayer partitioning; the potency tapers off at higher chain lengths, and eventually changes sign for the longest-chain alcohols, demonstrating an alcohol cutoff effect in a system that has no alcohol-binding pocket.     

A Steroid in a Lipid Bilayer: Localization, Orientation, and Energetics

Steroid hormones are known to freely partition into lipid bilayers. As a case study, we investigated the behavior of the steroid hormone cortisone in a model lipid bilayer. First, we looked at energy barriers involved in the partitioning of a single molecule into a bilayer using umbrella sampling molecular dynamics simulations. A rather wide well of −4.5 kcal/mol was observed in the interfacial region between the lipid headgroup and tailgroup. Next, using two unconstrained molecular dynamics simulations with cortisone initially positioned at distinct locations within a bilayer, we studied the preferred location and orientation of the molecule. Finally, we observed how cortisone molecules could spontaneously insert and localize in a bilayer from bulk solution. The three independent approaches produced a converged picture of how cortisone behaves in a model lipid bilayer.     

Small alcohols destabilize the KcsA tetramer via their effect on the membrane lateral pressure

Previously, it was shown that the tetrameric potassium channel KcsA when present in a lipid bilayer can be dissociated by trifluoroethanol [van den Brink-van der Laan, E., et al. (2004) Biochemistry 43, 4240-4250]. Here, we demonstrate that this is a general property of small alcohols. We found that small alcohols dissociate the KcsA tetramer, at a concentration that depends on their membrane affinity. Importantly, the efficiency of the alcohol-induced tetramer dissociation was found to correlate with the efficiency of both alcohol-induced bilayer leakage and acyl chain disordering. Our data suggest that the ability of small alcohols to induce KcsA tetramer dissociation and to function as anesthetics depends on their effect on the membrane lateral pressure.     

Accelerating membrane insertion of peripheral proteins with a novel membrane mimetic model

Characterizing atomic details of membrane binding of peripheral membrane proteins by molecular dynamics (MD) has been significantly hindered by the slow dynamics of membrane reorganization associated with the phenomena. To expedite lateral diffusion of lipid molecules without sacrificing the atomic details of such interactions, we have developed a novel membrane representation, to our knowledge, termed the highly mobile membrane-mimetic (HMMM) model to study binding and insertion of various molecular species into the membrane. The HMMM model takes advantage of an organic solvent layer to represent the hydrophobic core of the membrane and short-tailed phospholipids for the headgroup region. We demonstrate that using these components, bilayer structures are formed spontaneously and rapidly, regardless of the initial position and orientation of the lipids. In the HMMM membrane, lipid molecules exhibit one to two orders of magnitude enhancement in lateral diffusion. At the same time, the membrane atomic density profile of the headgroup region produced by the HMMM model is essentially identical to those obtained for full-membrane models, indicating the faithful representation of the membrane surface by the model. We demonstrate the efficiency of the model in capturing spontaneous binding and insertion of peripheral proteins by using the membrane anchor (γ-carboxyglutamic-acid-rich domain; GLA domain) of human coagulation factor VII as a test model. Achieving full insertion of the GLA domain consistently in 10 independent unbiased simulations within short simulation times clearly indicates the robustness of the HMMM model in capturing membrane association of peripheral proteins very efficiently and reproducibly. The HMMM model will provide significant improvements to the current all-atom models by accelerating lipid dynamics to examine protein-membrane interactions more efficiently.     

Hydration of polyethylene glycol-grafted liposomes.

This study aimed to characterize the effect of polyethylene glycol of 2000 molecular weight (PEG2000) attached to a dialkylphosphatidic acid (dihexadecylphosphatidyl (DHP)-PEG2000) on the hydration and thermodynamic stability of lipid assemblies. Differential scanning calorimetry, densitometry, and ultrasound velocity and absorption measurements were used for thermodynamic and hydrational characterization. Using a differential scanning calorimetry technique we showed that each molecule of PEG2000 binds 136 +/- 4 molecules of water. For PEG2000 covalently attached to the lipid molecules organized in micelles, the water binding increases to 210 +/- 6 water molecules. This demonstrates that the two different structural configurations of the PEG2000, a random coil in the case of the free PEG and a brush in the case of DHP-PEG2000 micelles, differ in their hydration level. Ultrasound absorption changes in liposomes reflect mainly the heterophase fluctuations and packing defects in the lipid bilayer. The PEG-induced excess ultrasound absorption of the lipid bilayer at 7.7 MHz for PEG-lipid concentrations over 5 mol % indicates the increase in the relaxation time of the headgroup rotation due to PEG-PEG interactions. The adiabatic compressibility (calculated from ultrasound velocity and density) of the lipid bilayer of the liposome increases monotonically with PEG-lipid concentration up to approximately 7 mol %, reflecting release of water from the lipid headgroup region. Elimination of this water, induced by grafted PEG, leads to a decrease in bilayer defects and enhanced lateral packing of the phospholipid acyl chains. We assume that the dehydration of the lipid headgroup region in conjunction with the increase of the hydration of the outer layer by grafting PEG in brush configuration are responsible for increasing thermodynamic stability of the liposomes at 5-7 mol % of PEG-lipid. At higher PEG-lipid concentrations, compressibility and partial volume of the lipid phase of the samples decrease. This reflects the increase in hydration of the lipid headgroup region (up to five additional water molecules per lipid molecule for 12 mol % PEG-lipid) and the weakening of the bilayer packing due to the lateral repulsion of PEG chains.     

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.     

Effect of changing the size of lipid headgroup on peptide insertion into membranes.

Adsorption of amphiphilic peptides to the headgroup region of a lipid bilayer is a common mode of protein-membrane interactions. Previous studies have shown that adsorption causes membrane thinning. The degree of the thinning depends on the degree of the lateral expansion caused by the peptide adsorption. If this simple molecular mechanism is correct, the degree of lateral expansion and consequently the membrane thinning should depend on the size of the headgroup relative to the cross section of the hydrocarbon chains. Previously we have established the connection between the alamethicin insertion transition and the membrane thinning effect. In this paper we use oriented circular dichroism to study the effect of varying the size of the headgroup, while maintaining a constant cross section of the lipid chains, on the insertion transition. A simple quantitative prediction agrees very well with the experiment.     

Interactions of cholesterol with lipid bilayers: the preferred configuration and fluctuations.

The free energy difference associated with the transfer of a single cholesterol molecule from the aqueous phase into a lipid bilayer depends on its final location, namely on its insertion depth and orientation within the bilayer. We calculated desolvation and lipid bilayer perturbation contributions to the water-to-membrane transfer free energy, thus allowing us to determine the most favorable location of cholesterol in the membrane and the extent of fluctuations around it. The electrostatic and nonpolar contributions to the solvation free energy were calculated using continuum solvent models. Lipid layer perturbations, resulting from both conformational restrictions of the lipid chains in the vicinity of the (rigid) cholesterol backbone and from cholesterol-induced elastic deformations, were calculated using a simple director model and elasticity theory, respectively. As expected from the amphipathic nature of cholesterol and in agreement with the available experimental data, our results show that at the energetically favorable state, cholesterol's hydrophobic core is buried within the hydrocarbon region of the bilayer. At this state, cholesterol spans approximately one leaflet of the membrane, with its OH group protruding into the polar (headgroup) region of the bilayer, thus avoiding an electrostatic desolvation penalty. We found that the transfer of cholesterol into a membrane is mainly driven by the favorable nonpolar contributions to the solvation free energy, whereas only a small opposing contribution is caused by conformational restrictions of the lipid chains. Our calculations also predict a strong tendency of the lipid layer to elastically respond to (thermally excited) vertical fluctuations of cholesterol so as to fully match the hydrophobic height of the solute. However, orientational fluctuations of cholesterol were found to be accompanied by both an elastic adjustment of the surrounding lipids and by a partial exposure of the hydrophobic cholesterol backbone to the polar (headgroup) environment. Our calculations of the molecular order parameter, which reflects the extent of orientational fluctuations of cholesterol in the membrane, are in good agreement with available experimental data.     

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.     

A Molecular Dynamics Investigation of Lipid Bilayer Perturbation by PIP2

Phosphoinositides like phosphatidylinositol 4,5-bisphosphate (PIP₂) are negatively charged lipids that play a pivotal role in membrane trafficking, signal transduction, and protein anchoring. We have designed a force field for the PIP₂ headgroup using quantum mechanical methods and characterized its properties inside a lipid bilayer using molecular dynamics simulations. Macroscopic properties such as area/headgroup, density profiles, and lipid order parameters calculated from these simulations agree well with the experimental values. However, microscopically, the PIP₂ introduces a local perturbation of the lipid bilayer. The average PIP₂ headgroup orientation of 45° relative to the bilayer normal induces a unique, distance-dependent organization of the lipids that surround PIP₂. The headgroups of these lipids preferentially orient closer to the bilayer normal. This perturbation creates a PIP₂ lipid microdomain with the neighboring lipids. We propose that the PIP₂ lipid microdomain enables the PIP₂ to function as a membrane-bound anchoring molecule.     

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.