Chemically induced phospholipid translocation across biological membranes

Chemical means of manipulating the distribution of lipids across biological membranes is of considerable interest for many biomedical applications as a characteristic lipid distribution is vital for numerous cellular functions. Here we employ atomic-scale molecular simulations to shed light on the ability of certain amphiphilic compounds to promote lipid translocation (flip-flops) across membranes. We show that chemically induced lipid flip-flops are most likely pore-mediated: the actual flip-flop event is a very fast process (time scales of tens of nanoseconds) once a transient water defect has been induced by the amphiphilic chemical (dimethylsulfoxide in this instance). Our findings are consistent with available experimental observations and further emphasize the importance of transient membrane defects for chemical control of lipid distribution across cell membranes.     

Pore formation coupled to ion transport through lipid membranes as induced by transmembrane ionic charge imbalance: atomistic molecular dynamics study

We have employed atomic-scale molecular dynamics simulations to address ion transport through transient water pores in phospholipid membranes. The formation of a water pore is induced by a transmembrane ionic charge imbalance, which gives rise to a significant potential difference across the membrane. The subsequent transport of ions through the pore discharges the transmembrane potential and makes the water pore metastable, leading eventually to its sealing. The findings highlight the importance of ionic charge fluctuations in spontaneous pore formation and their role in ion leakage through protein-free lipid membranes.     

Cation and anion transport through hydrophilic pores in lipid bilayers

To understand the origin of transmembrane potentials, formation of transient pores, and the movement of anions and cations across lipid membranes, we have performed systematic atomistic molecular dynamics simulations of palmitoyl-oleoyl-phosphatidylcholine (POPC) lipids. A double bilayer setup was employed and different transmembrane potentials were generated by varying the anion (Cl-) and cation (Na+) concentrations in the two water compartments. A transmembrane potential of approximately 350 mV was thereby generated per bilayer for a unit charge imbalance. For transmembrane potential differences of up to approximately 1.4 V, the bilayers were stable, over the time scale of the simulations (10-50 ns). At larger imposed potential differences, one of the two bilayers breaks down through formation of a water pore, leading to both anion and cation translocations through the pore. The anions typically have a short residence time inside the pore, while the cations show a wider range of residence times depending on whether they bind to a lipid molecule or not. Over the time scale of the simulations, we do not observe the discharge of the entire potential difference, nor do we observe pore closing, although we observe that the size of the pore decreases as more ions translocate. We also observed a rare lipid flip-flop, in which a lipid molecule translocated from one bilayer leaflet to the opposite leaflet, assisted by the water pore.     

Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) is a small amphiphilic molecule which is widely employed in cell biology as an effective penetration enhancer, cell fusogen, and cryoprotectant. Despite the vast number of experimental studies, the molecular basis of its action on lipid membranes is still obscure. A recent simulation study employing coarse-grained models has suggested that DMSO induces pores in the membrane (Notman, R.; Noro, M.; O'Malley, B.; Anwar, J. J. Am. Chem. Soc. 2006, 128, 13982-13983). We report here the molecular mechanism for DMSO's interaction with phospholipid membranes ascertained from atomic-scale molecular dynamics simulations. DMSO is observed to exhibit three distinct modes of action, each over a different concentration range. At low concentrations, DMSO induces membrane thinning and increases fluidity of the membrane's hydrophobic core. At higher concentrations, DMSO induces transient water pores into the membrane. At still higher concentrations, individual lipid molecules are desorbed from the membrane followed by disintegration of the bilayer structure. The study provides further evidence that a key aspect of DMSO's mechanism of action is pore formation, which explains the significant enhancement in permeability of membranes to hydrophilic molecules by DMSO as well as DMSO's cryoprotectant activity. The reduction in the rigidity and the general disruption of the membrane induced by DMSO are considered to be prerequisites for membrane fusion processes. The findings also indicate that the choice of DMSO concentration for a given application is critical, as the concentration defines the specific mode of the solvent's action. Knowledge of the distinct modes of action of DMSO and associated concentration dependency should enable optimization of current application protocols on a rational basis and also promote new applications for DMSO.     

Ion transport through chemically induced pores in protein-free phospholipid membranes

We address the possibility of being able to induce the trafficking of salt ions and other solutes across cell membranes without the use of specific protein-based transporters or pumps. On the basis of realistic atomic-scale molecular dynamics simulations, we demonstrate that transmembrane ionic leakage can be initiated by chemical means, in this instance through addition of dimethyl sulfoxide (DMSO), a solvent widely used in cell biology. Our results provide compelling evidence that the small amphiphilic solute DMSO is able to induce transient defects (water pores) in membranes and to promote a subsequent diffusive pore-mediated transport of salt ions. The findings are consistent with available experimental data and offer a molecular-level explanation for the experimentally observed activities of DMSO solvent as an efficient penetration enhancer and a cryoprotectant, as well as an analgesic. Our findings suggest that transient pore formation by chemical means could emerge as an important general principle for therapeutics.     

Phosphate-mediated arginine insertion into lipid membranes and pore formation by a cationic membrane peptide from solid-state NMR

The insertion of charged amino acid residues into the hydrophobic part of lipid bilayers is energetically unfavorable yet found in many cationic membrane peptides and protein domains. To understand the mechanism of this translocation, we measured the (13)C-(31)P distances for an Arg-rich beta-hairpin antimicrobial peptide, PG-1, in the lipid membrane using solid-state NMR. Four residues, including two Arg's, scattered through the peptide were chosen for the distance measurements. Surprisingly, all residues show short distances to the lipid (31)P: 4.0-6.5 A in anionic POPE/POPG membranes and 6.5-8.0 A in zwitterionic POPC membranes. The shortest distance of 4.0 A, found for a guanidinium Czeta at the beta-turn, suggests N-H...O-P hydrogen bond formation. Torsion angle measurements of the two Arg's quantitatively confirm that the peptide adopts a beta-hairpin conformation in the lipid bilayer, and gel-phase 1H spin diffusion from water to the peptide indicates that PG-1 remains transmembrane in the gel phase of the membrane. For this transmembrane beta-hairpin peptide to have short (13)C-(31)P distances for multiple residues in the molecule, some phosphate groups must be embedded in the hydrophobic part of the membrane, with the local (31)P plane parallel to the beta-strand. This provides direct evidence for toroidal pores, where some lipid molecules change their orientation to merge the two monolayers. We propose that the driving force for this toroidal pore formation is guanidinium-phosphate complexation, where the cationic Arg residues drag the anionic phosphate groups along as they insert into the hydrophobic part of the membrane. This phosphate-mediated translocation of guanidinium ions may underlie the activity of other Arg-rich antimocrobial peptides and may be common among cationic membrane proteins.     

Replacing the cholesterol hydroxyl group with the ketone group facilitates sterol flip-flop and promotes membrane fluidity

The 3alpha-hydroxyl group is a characteristic structural element of all membrane sterol molecules, while the 3-ketone group is more typically found in steroid hormones. In this work, we investigate the effect of substituting the hydroxyl group in cholesterol with the ketone group to produce ketosterone. Extensive atomistic molecular dynamics simulations of saturated lipid membranes with either cholesterol or ketosterone show that, like cholesterol, ketosterone increases membrane order and induces condensation. However, the effect of ketosterone on membrane properties is considerably weaker than that of cholesterol. This is largely due to the unstable positioning of ketosterone at the membrane-water interface, which gives rise to a small but significant number of flip-flop transitions, where ketosterone is exchanged between membrane leaflets. This is remarkable, as flip-flop motions of sterol molecules have not been previously reported in analogous lipid bilayer simulations. In the same context, ketosterone is found to be more tilted with respect to the membrane normal than cholesterol. The atomic level mechanism responsible for the increase of the steroid tilt and the promotion of flip-flops is the decrease in polar interactions at the membrane-water interface. Interactions between lipids or water and the ketone group are found to be weaker than in the case of the hydroxyl group, which allows ketosterone to penetrate through the hydrocarbon region of a membrane.     

Factors influencing the transport of short-chain alcohols through mesoporous gamma-alumina membranes

The pressure-driven transport of water, ethanol, and 1-propanol through supported gamma-alumina membranes with different pore diameters is reported. Water and alcohols had similar permeabilities when they were transported through gamma-alumina membranes with average pore diameters of 4.4 and 6.0 nm, and the permeability coefficient was found to be proportional to the square of pore size, in accordance with a viscous flow mechanism. For transport through membranes with an average diameter of 3.2 nm, the behavior of water was in accordance with the viscous flow mechanism, but the permeability of the membrane for ethanol and 1-propanol was much smaller than expected and could not be explained in terms of viscous flow. Although the low permeability of the membrane with 3.2 nm pores for ethanol and 1-propanol was partly due to the presence of small amounts of water in the alcohols, the permeability coefficients were still substantially smaller when water was absent. This intrinsic difference between water and alcohol may be due to differences in molecular size, chemisorption of alcohols on the oxide pore wall, which would lead to a reduction of the effective pore size, and/or a certain degree of translational ordering of the alcohol molecules inside the membrane pores, which leads to an effectively higher viscosity and, therefore, to a higher transport resistance.     

The effect of lipid oxidation on the water permeability of phospholipids bilayers

The effect of lipid oxidation on water permeability of phosphatidylcholine membranes was investigated by means of both scattering stopped flow experiments and atomistic molecular dynamics simulations. Formation of water pores followed by a significant enhancement of water permeability was observed. The molecules of oxidized phospholipids facilitate pore formation and subsequently stabilize water in the membrane interior. A wide range of oxidation ratios, from 15 to 100 mol%, was considered. The degree of oxidation was found to strongly influence the time needed for the opening of a pore. In simulations, the oxidation ratio of 75 mol% was found to be a threshold for spontaneous pore formation in the tens of nanosecond timescale, whereas 15 mol% of oxidation led to significant water permeation in the timescale of seconds. Once a pore was formed, the water permeability was found to be virtually independent of the oxidation ratio.     

Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion

Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.     

The analytical potential of chemoreception at bilayer lipid membranes

The potential use of the bilayer lipid membrane as an electrochemical sensor is discussed through a study of model systems known to cause increased membrane conductance. The limit of detection for amphotericin B, a molecule capable of forming membrane pores, is in the region of 1O^-9 M. The current—time profile is discussed in terms of a mechanism which involves micelle formation in the aqueous and lipid phases. Unlike previous experiments, two current maxima with time are observed for valinomycin response (limit of detection 1O^-11 M). The first transient signal is attributed to increased membrane permeability caused by a conformational change in valinomycin in the “surface” volume of the bilayer. Selective interactions at membranes and the nature of membrane responses are discussed in terms of analytical parameters.     

Molecular ferries: membrane carriers that promote phospholipid flip-flop and chloride transport

The facilitated transport of ionic or polar solutes through biological membranes is an essential process for cellular life, and a major technical goal of the pharmaceutical industry. Synthetic receptors with affinities for anions are shown to act as molecular ferries and facilitate the movement of chloride ions and salts across vesicle and cell membranes. A process that competes with chloride transport is phospholipid translocation or flip-flop. This has led to the development of synthetic scramblases that can alter the transmembrane distribution of phospholipids and induce biological responses such as membrane enzyme activation. The facilitated translocation of phospholipids with multiply-charged head groups, like phosphatidylserine, is a difficult supramolecular challenge that requires a complementary, multitopic receptor with appropriate amphiphilicity.     

Pore spanning lipid bilayers on mesoporous silica having varying pore size

Synthetic lipid bilayers have similar properties as cell membranes and have been shown to be of great use in the development of novel biomimicry devices. In this study, lipid bilayer formation on mesoporous silica of varying pore size, 2, 4, and 6 nm, has been investigated using quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescent recovery after photo bleaching (FRAP), and atomic force microscopy (AFM). The results show that pore-spanning lipid bilayers were successfully formed regardless of pore size. However, the mechanism of the bilayer formation was dependent on the pore size, and lower surface coverages of adsorbed lipid vesicles were required on the surface having the smallest pores. A similar trend was observed for the lateral diffusion coefficient (D) of fluorescently labeled lipid molecules in the membrane, which was lowest on the surface having the smallest pores and increased with the pore size. All of the pore size dependent observations are suggested to be due to the hydrophilicity of the surface, which decreases with increased pore size.     

Demulsification of water-in-oil emulsions via filtration through a hydrophilic polymer membrane

It is known that hydrophobic microfiltration membranes can be used for demulsification of oil-in-water (o/w) emulsion due to coalescence of oil droplets in membrane pores. This study demonstrates that a hydrophilic polymer membrane can be used for the demulsification of surfactant-stabilized water-in-oil (w/o) emulsions. The success of demulsification is dependent on the type of emulsions and membrane used. Membrane pore size and transmembrane pressure were found to affect demulsification efficiency (DM), while other factors, such as membrane thickness and initial water content have slight or almost no effect. A coalescence mechanism of the demulsification phenomenon is also discussed. The separation process is not based on sieving effects due to a difference in membrane pore size, but is determined by droplet interactions with membrane surface.     

Pore blocking mechanisms during early stages of membrane fouling by colloids

A method based on a simple linear regression fitting was proposed and used to determine the type, the chronological sequence, and the relative importance of individual fouling mechanisms in experiments on the dead-end filtration of colloidal suspensions with membranes ranging from loose ultrafiltration (UF) to nanofiltration (NF) to non-porous reverse osmosis (RO). For all membranes, flux decline was consistent with one or more pore blocking mechanisms during the earlier stages and with the cake filtration mechanism during the later stages of filtration. For ultrafiltration membranes, pore blocking was identified as the largest contributor to the observed flux decline. The chronological sequence of blocking mechanisms was interpreted to depend on the size distribution and surface density of membrane pores. For salt-rejecting membranes, the flux decline during the earlier stages of filtration was attributed to either intermediate blocking of relatively more permeable areas of the membrane skin, or to the cake filtration in its early transient stages, or a combination of these two mechanisms. The findings emphasize the practical importance of the clear identification of, and differentiation between mechanisms of pore blocking and cake formation as determining the potential for the irreversible fouling of membranes and the efficiency of membrane cleaning.     

Lipids out of equilibrium: energetics of desorption and pore mediated flip-flop

The potential of mean force (PMF) of a phospholipid in a bilayer is a key thermodynamic property that describes the energetic cost of localized lipid defects. We have calculated the PMF by umbrella sampling using molecular dynamics simulations. The profile has a deep minimum at the equilibrium position in the bilayer and steeply rises for displacements both deeper into the bilayer and moving away from the bilayer. As the lipid loses contact with the bilayer, the profile abruptly flattens without a significant barrier. The calculated free energy difference of 80 kJ/mol between the minimum of the PMF and the value in water agrees well with the free energy difference calculated from the experimentally measured critical micelle concentration. Significant water/lipid defects form when a lipid is forced into the bilayer interior, in the form of a small water pore that spans the membrane. The energy required to form such a water pore is also found to be 80 kJ/mol. On the basis of this energy, we estimate the lipid flip-flop rate and permeability rate of sodium ions. The resulting rates are in good agreement with experimental measurements, suggesting lipid flip-flop and basal permeability of ions are pore mediated.     

Modeling peptide binding to anionic membrane pores

Peptide‐induced pore formation in membranes can be dissected into two steps: pore formation and peptide binding to the pore. A computational method is proposed to study the second step in anionic membranes. The electrostatic potential is obtained from numerical solutions to the Poisson–Boltzmann equation and is then used in conjunction with IMM1 (implicit membrane model 1). A double charge layer model is used to incorporate the effects of the membrane dipole potential. Inhomogeneity of the charge density in the pore, characterized by explicit membrane simulations of toroidal pores, is included in the model. This approach was applied to two extensively studied peptides, magainin and melittin. In agreement with previous work, binding to toroidal pores is more favorable than binding to the flat membrane. The dependence of binding energy on anionic content exhibits different patterns for the two peptides, in correlation with the different lipid selectivity that has been observed experimentally. © 2013 Wiley Periodicals, Inc.