Biomembrane Systems Research Articles

Molecular dynamics simulations of membrane proteins under asymmetric ionic concentrations

A computational method is developed to allow molecular dynamics simulations of biomembrane systems under realistic ionic gradients and asymmetric salt concentrations while maintaining the conventional periodic boundary conditions required to minimize finite-size effects in an all-atom explicit solvent representation. The method, which consists of introducing a nonperiodic energy step acting on the ionic species at the edge of the simulation cell, is first tested with illustrative applications to a simple membrane slab model and a phospholipid membrane bilayer. The nonperiodic energy-step method is then used to calculate the reversal potential of the bacterial porin OmpF, a large cation-specific β-barrel channel, by simulating the I-V curve under an asymmetric 10:1 KCl concentration gradient. The calculated reversal potential of 28.6 mV is found to be in excellent agreement with the values of 26–27 mV measured from lipid bilayer experiments, thereby demonstrating that the method allows realistic simulations of nonequilibrium membrane transport with quantitative accuracy. As a final example, the pore domain of Kv1.2, a highly selective voltage-activated K+ channel, is simulated in a lipid bilayer under conditions that recreate, for the first time, the physiological K+ and Na+ concentration gradients and the electrostatic potential difference of living cells.

Bridging the gap between microscopic and macroscopic views of air/aqueous salt interfaces

Physico-chemical phenomena in atmospheric aerosol, geochemical, and biomembrane systems are strongly influenced by interfacial ion distributions, and the resulting interfacial electrostatic fields. Results from early surface potentiometry and from recent phase-resolved (phase-sensitive (PS-) and heterodyne-detected (HD-)) vibrational sum frequency generation (VSFG) spectroscopy measurements on aqueous salt solutions have been interpreted in terms of anions and cations being distributed within the interfacial region according to their surface propensity, forming an ionic double layer. This molecular-level picture has been consistent with the distribution (density) profiles obtained by MD simulations of aqueous interfaces of halide salts. Here we discuss PS- and HD-VSFG results revealing that for some oxyanion- and Mg2+-based salts, the interpretation of the electric field in terms of determining ion surface propensity and/or charge could be misleading. We also discuss the intricacies of counterion effects and the correlations with surface potential versus phase-resolved (PS- or HD-) VSFG measurements.

Interactions of a cationic surfactant – (benzyloxymethyl) dodecyldimethylammonium chloride with model biomembrane systems

Phospholipids are the main components of biological membranes. The aim of the present study was to determine the influence of a cationic surfactant on phospholipid structure and dynamics. Fourier transform infrared (FTIR) and dielectric relaxation (DRS) spectroscopies as well as small-angle X-ray scattering (SAXS) with synchrotron radiation have been used to analyse the structure of fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in the presence of a quaternary ammonium surfactant: (benzyloxymethyl) dodecyldimethylammoniumchloride (BzMDDAC). The presence of the surfactant caused changes in the temperature of the DMPC phase transition, as revealed using FTIR and DRS measurements. This change results from the disappearance of the multilamellar phase of DMPC and the formation of the unilamellar (most likely bicellar) phase, as indicated by the SAXS results.

Gibbs energy determinants of lipoprotein insertion into lipid membranes: the case study of Ras proteins

In a combined chemical-biological and biophysical approach we explored the Gibbs (free) energy contributions to the membrane partitioning of lipidated proteins, and compared the theoretical predictions with recent experimental data on the membrane insertion of Ras proteins of various anchor systems into rationally designed model biomembrane systems. Various factors fostering or reducing the membrane partitioning properties are discussed, including hydrophobic effects, lipid chain mismatch, electrostatic interactions, membrane-mediated protein-protein interactions, and terms that account for line tension effects between coexisting lipid domains, lipid sorting, and changes in the lateral organization of the lipid bilayer system. From these data, it is apparent that two membrane anchoring motifs are needed to facilitate firm membrane binding. For heterogeneous membranes, localization and sequestration at domain boundaries as well as formation of protein clusters and collective lateral organization via an effective lipid sorting mechanism provide complementary ways of inducing membrane nanodomains that could potentially operate as effective, high fidelity signalling platforms.

Multidimensional Assembly of S-Layer Proteins on Mobility-Controlled Polyelectrolyte Multilayers.

Polyelectrolyte multilayers have been vastly utilized as an assembling platform for various biomaterials because of their soft and charged surface characteristics, analogous to biomembrane systems. In particular, polyelectrolyte chains with high self-diffusivity can effectively transfer the surface mobility to the assembling biomolecular species, facilitating the ordered self-assembly. Herein, highly diffusional cationic polyelectrolyte chains of linear polyethylenimine are employed to induce direct binding with negatively charged bacterial surface layer proteins, which eventually lead to large-scale two-dimensional crystals. Notably, at the elevated incubation temperature, a transitory intermediate of one-dimensional chain structure is observed. We reveal that this one-dimensional intermediate is a stable precursor toward two-dimensional crystal arrays.

Detection of lipid raft domains in neutral and anionic Langmuir monolayers and bilayers of complex lipid composition

In reference to the complexity and heterogeneity of cellular membranes, the structure and lateral ordering of lipid monolayers and bilayers composed of multi-component lipid mixtures have been investigated and compared in the present study. These complex model biomembrane systems represent valuable model systems, e.g. for studies of lipid–peptide interactions, where an integrated in situ multi-technique approach using both monolayer and bilayer techniques is required. A zwitterionic (3 components) and an anionic (5 components) heterogeneous model membrane system have been characterized that consist of saturated and unsaturated phospholipids as well as cholesterol. Lipid monolayers were analyzed by surface X-ray scattering techniques, and both the vertical structure (electron density profile) and the degree of in-plane ordering were determined as a function of surface pressure and temperature. The corresponding structure and lateral organisation of the bilayer membranes were characterized by atomic force microscopy. Both monolayers and bilayers reveal ordered domain formation, the monolayer ones being of much smaller size and different temperature stability, however. Furthermore, the charge density of the lipid monolayer has a drastic influence on the size of ordered domains as well as the intermolecular distances of the lipid molecules.

Characterization of the Dynamical Properties of Ras Proteins upon Insertion into Model Biomembrane Systems

Proteins of the Ras superfamily are located at the inner leaflet of the plasma membrane and play an important role in cellular signal transduction processes, such as cell growth or differentiation. Since nanoclustering is discussed as an effective signaling mechanism, determination of the rotational and translational dynamics of membrane-associated Ras proteins may help revealing additional mechanistic information about protein clustering and interactions with effector proteins. In this study, different biophysical approaches are combined to investigate the partitioning behavior and dynamical properties of two Ras isoforms, namely N- and K-Ras, in unilamellar vesicles of varied lipid composition. As model biomembrane systems, pure fluid-like (liquid-disordered), neutral and anionic heterogeneous model raft membranes were used. Confocal laser scanning microscopy was applied to gain information on the partitioning of the Ras lipoproteins into the different model membrane systems. Fluorescence anisotropy and fluorescence correlation spectroscopy experiments were carried out to yield information on dynamical properties, such as the rotational correlation time and the translational diffusion coefficient, of the fully lipidated BODIPY-labeled Ras proteins. The results reveal a preferential incorporation of Ras into fluid-like liquid-disordered lipid domains, independent of the Ras isoform and GDP/GTP-loading [1]. Although the rotational mobility remains quite high upon membrane insertion, the translational dynamics is limited by the viscosity of the respective lipid system, in agreement with results obtained in in vivo studies. Depending on the nucleotide loading, no significant differences could be detected with respect to the dynamics upon membrane incorporation.Reference[1] Weise K, Kapoor S, Denter C, Nikolaus J, Opitz N, Koch S, Triola G, Herrmann A, Waldmann H, and Winter R (2011) J. Am. Chem. Soc. 133:880-887.

Biosorption processes in biomembrane systems during water treatment of surface water sources

The characteristics of a system with bioactive powdered activated carbon and microfiltration have been studied under conditions of the aerobic treatment of natural water from the water storages of Guan Ting (China), the Moskva River, and the Yauza River (Russia). The removal of organic matter in the system was estimated in terms of the permanganate oxidizability and UV absorption at λ = 254 nm (UV254) and λ = 410 nm (UV410). The average removal efficiency amounted to 68.42, 75.61, and 87.50%, respectively, at water temperature 10°C. The water treatment process (at 20°C) began immediately after the start-up of the plant at the expense of the adsorption on activated carbon that guaranteed a high speed of removal of organic pollutants in the absence of mature microflora. By the time the adsorption capacity of carbon was exhausted, the microflora was able to mature ensuring in combination with the powdered activated carbon a high speed of removal. In order to guarantee the biological stability of water, the removal degree of assimilable organic carbon amounted to 60.2% and the purified water met the requirements of the recommended criterion (100 mg acetate-C/dm3).

Effects of temperature and pressure on the lateral organization of model membranes with functionally reconstituted multidrug transporter LmrA

To contribute to the understanding of membrane protein function upon application of pressure, we investigated the influence of hydrostatic pressure on the conformational order and phase behavior of the multidrug transporter LmrA in biomembrane systems. To this end, the membrane protein was reconstituted into various lipid bilayer systems of different chain length, conformation, phase state and heterogeneity, including raft model mixtures as well as some natural lipid extracts. In the first step, we determined the temperature stability of the protein itself and verified its reconstitution into the lipid bilayer systems using CD spectroscopic and AFM measurements, respectively. Then, to yield information on the temperature and pressure dependent conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined, which report on the conformation and phase state of the lipid bilayer system. The temperature-dependent measurements were carried out in the temperature range 5–70 °C, and the pressure dependent measurements were performed in the range 1–200 MPa. The data show that the effect of the LmrA reconstitution on the conformation and phase state of the lipid matrix depends on the fluidity and hydrophobic matching conditions of the lipid system. The effect is most pronounced for fluid DMPC and DMPC with low cholesterol levels, but minor for longer-chain fluid phospholipids such as DOPC and model raft mixtures such as DOPC/DPPC/cholesterol. The latter have the additional advantage of using lipid sorting to avoid substantial hydrophobic mismatch. Notably, the most drastic effect was observed for the neutral/glycolipid natural lipid mixture. In this case, the impact of LmrA incorporation on the increase of the conformational order of the lipid membrane was most pronounced. As a consequence, the membrane reaches a mechanical stability which makes it very insensitive to application of pressures as high as 200 MPa. The results are correlated with the functional properties of LmrA in these various lipid environments and upon application of high hydrostatic pressure and are discussed in the context of other work on pressure effects on membrane protein systems.

Kinetics and mechanisms of pressure-induced phase transitions of ternary model biomembrane systems

Kinetics and mechanisms of pressure-induced phase transitions of ternary model biomembrane systems

Functional Determinants of a Synthetic Vesicle Fusion System

Selective membrane mergers may be driven by small-molecule recognition between synthetic surface-displayed fusogens which bear vancomycin glycopeptide and its native binding target, D-Ala-D-Ala dipeptide. These recognition motifs are membrane anchored by antimicrobial peptide magainin II and a phosphatidylethanolamine lipid derivative, respectively. We report herein characterization of this synthetic membrane fusion reaction with regard to the following: effects of fusogen concentration, lipid composition, and membrane charge. Our findings indicate that these parameters are determinants of fusion rate, vesicle stability, peptide binding, catalytic fusion and membrane disruption during fusion. Notably, these data indicate the importance of coupling between molecular recognition and insertion for bilayer activation as well as the critical role of membrane subdomain formation for membrane fusion reactivity. These phenomena are general to lipid membrane chemistry, and therefore these findings provide a guideline for understanding more complex biomembrane systems.

Towards an Understanding of the Temperature/ Pressure Configurational and Free-Energy Landscape of Biomolecules

Besides temperature, we used hydrostatic pressure as a physical parameter for studying the stability and energetics of biomolecular systems. High pressure is also an important feature of certain natural environments, and the high-pressure phase behaviour of biomolecules is of increasing biotechnological interest. By using thermodynamic, spectroscopic, and scattering techniques, the temperature and pressure-dependent structure and phase behaviour of model biomembrane systems and proteins have been studied. The results demonstrate that combined temperature–pressure-dependent studies can help delineate the conformational and free-energy landscape of biomolecules and hence help to elucidate which features and thermodynamic parameters are essential in determining their stability. Moreover, we present data on the effect of various types of cosolvents on the temperature- and pressure-dependent structure and stability of proteins. We also introduce pressure as a kinetic variable. Applying the pressure-jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction and spectroscopic techniques, the kinetics of lipid phase transitions and un/refolding reactions of proteins have been studied. Finally, recent advances in using the pressure axis for studying misfolding, aggregation, and amyloid fibril formation of proteins and its potential biotechnological and biomedical relevance will be discussed. We conclude with an outlook of the perspectives of high pressure research.

Free radical scavenging and antioxidative activities of caffeic acid phenethyl ester (CAPE) and its related compounds in solution and membranes: A structure–activity insight

Caffeic acid phenethyl ester (CAPE) together with caffeic acid, ferulic acid and ethyl ferulate are constituents of propolis and structurally related, which allowed us to gather important information regarding the structure–activity relationships underlying the biological activity of such compounds. In this work, we have investigated the direct scavenging effects of the antioxidants on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, galvinoxyl radicals and superoxide anions ( O 2 - ) , as well as the anti-lipoperoxidative capacity on human erythrocytes hemolysis and rat liver microsomal membranes peroxidation induced in vitro by two different sources of free radicals: 2,2′-azobis(2-amidinopropane) (AAPH) and Fe 2+/ascorbate (OH ). The results established that the radical scavenging activity of the compounds increased with increasing numbers of hydroxyl groups or catechol moieties in the molecule, while in the biomembrane systems, the antioxidative activity of the test compounds depends not only on the hydroxyl groups or catechol rings but also on the polarity and hydrophobicity of the antioxidants. In addition, CAPE is the most effective antioxidant of the compounds we tested in our in vitro systems.

Characterization of Proton Transport across a Waveguide-Supported Lipid Bilayer

Cellular energy transduction processes are often driven by transmembrane ion gradients, and numerous artificial biomembrane systems have been developed that allow for chemically or light-induced charge transport into/out of liposomes. Liposomal architectures, however, are not readily interfaced to a solid-state transducer. Formation of an ion gradient across a planar-supported membrane, "wired" to a substrate electrode, may ultimately allow utilization of the potential energy to drive other electrochemical processes. Described here is a novel conductive polymer/planar waveguide assembly that provides for highly sensitive transduction of proton transport across a planar-supported lipid bilayer (PSLB). A quinone proton shuttle is embedded in the PSLB, which is coupled to the planar optical waveguide electrode through a pH-sensitive, self-assembled conductive polymer film. Interfacial potential and absorbance changes in the conductive polymer film provide for sensitive characterization of transmembrane proton transport. The general and flexible nature of this architecture makes it adaptable to many different types of transmembrane transport chemistries, particularly light-activated systems.

Low-frequency critical dynamics in (CnH2n+1NH3)2SnCl6 model biomembrane systems

Abstract Nuclear magnetic resonance has been employed as a probe for the collective hydrocarbon chain dynamics in the organic–inorganic model biomembranes (CnH2n+1NH3)2SnCl6, undergoing order–disorder and conformational phase transitions. No anomalies were observed in the laboratory-frame spin–lattice relaxation measurements at the order–disorder phase transitions, whereas a discontinuity was manifest at the conformational phase transitions characteristic of a first-order phase transition. On the other hand, our rotating frame spin–lattice relaxation measurements revealed a low-frequency critical collective chain dynamics in the kilohertz regime associated with the order–disorder phase transition.

Investigation of artificial biomembrane systems in biopartitioning micellar chromatography by method of mathematical design

An attempt to create and study an artificial membrane system was realized via biopartitioning micellar liquid chromatography. Towards this end the known formula of membrane permeability (on the basis of Fick's diffusion equation) was modified so that membrane permeability may be estimated in terms of chromatographic characteristics. The two-factoral experiments on the basis of mathematical design of second order were carried out. The regression equations are derived which describe the dependence of membrane permeability on the concentration of polyoxyethylene (23) lauryl ether in the mobile phase and its flow-rate for compounds with biomedical significance. Some regularities were revealed, which characterize the permeability of compounds of the different nature through membranes. The extremal dependence (with passing through minimum) of permeability on the concentration of non-ionic surfactant was observed for anionic compounds. The increasing character of permeability in relation with flow-rate of mobile phase was recognized for cationic samples. Both dependences were basically fulfilled for zwitterionic compounds.

Molecular dynamics simulations of ionic concentration gradients across model bilayers

To model a concentration gradient across a biomembrane, we have performed all-atom molecular dynamics simulations of NaCl solutions separated by two oppositely charged plates. We have employed the recently formulated three-dimensional Ewald summation with correction (EW3DC) technique for calculations of long-range electrostatics in two-dimensionally periodic systems, allowing for different salt concentrations on the two sides of the plates. Six simulations were run, varying the salt concentrations and plate surface charge density in a biologically relevant range. The simulations reveal well-defined, atomic-level asymmetries between the two sides: distinct translational and rotational orderings of water molecules; differing ion residency times; a clear wetting layer adjacent only to the negative plate; and marked differences in charge density/potential profiles which reflect the microscopic behavior. These phenomena, which may play important roles in membrane and ion channel physiology, result primarily from the electrostatics and asymmetry of water molecules, and not from the salt ions. In order to establish that EW3DC can accurately capture fundamental electrostatic interactions important to asymmetric biomembrane systems, the CHARMM force-field (with the corrected Ewald sum) has been used. Comparison of the results with previously published simulations of electrolyte near charged surfaces, which employed different force-fields, shows the robustness of the CHARMM potential and gives confidence in future all-atom bilayer simulations using EW3DC and CHARMM.

Computer simulation studies of model biological membranes.

This Account is focused on computer simulation studies of model biological membrane systems with potential applications in biomedical research. In the past decade, classical molecular dynamics has provided novel insights into the properties of model biomembrane systems, including the nature of the DNA-lipid interactions, the effect of pore-forming transmembrane peptides on the lipid environment, and the partitioning of volatile anesthetic molecules. Such simulations, employing full atomic detail, are typically restricted to systems of dimensions less than approximately 10 nm. Simplified models of the coarse-grain type have been intended to bridge the gap between full atomistic detail and the mesoscopic (micron) regime. The use of such models is illustrated with the example of anesthetics in a phospholipid bilayer.

Plasmon resonance spectroscopy: probing molecular interactions at surfaces and interfaces

Surface plasmon resonance (SPR) spectroscopy can be applied to a wide variety of interfacial systems. It involves resonant excitation by polarized light of electronic oscillations (plasmons) in a thin metal film. These generate a surface‒localized evanescent electromagnetic field that can be used to probe the optical properties perpendicular to the film plane of materials immobilized at the surface. Spectra depend on three parameters: refractive index (n), absorption coefficient (k) and thickness (t). Maxwell′s equations provide an analytical relationship between these properties and SPR spectra, allowing their evaluation. An extension of this methodology, called coupled plasmon‒waveguide resonance (CPWR or PWR), is able to characterize film properties both perpendicular and parallel to the surface plane. In a PWR device, the metal film is covered with a dielectric coating that acts as an optical amplifier, provides protection for the metal layer, and possesses a surface that allows various molecular immobilization strategies. The exceptionally narrow line widths of PWR spectra yield enhanced sensitivity and resolution. The application of this technology to several biomembrane systems will be described, demonstrating its ability to observe both binding and structural events occurring during membrane protein function.

Pressure effects on the structure of lyotropic lipid mesophases and model biomembrane systems

Lipid systems, which provide valuable model systems for biological membranes, display a variety of polymorphic phases, depending on their molecular structure and environmental conditions. By use of X-ray and neutron diffraction the temperature- and pressure-dependent structure and phase behavior of lipid systems, differing in chain configuration and headgroup structure, have been studied. Besides lamellar phases also nonlamellar phases have been investigated. Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of lyotropic lipid mesophases, but also because high pressure is an important feature of certain natural membrane environments (e.g., marine biotopes) and because the high pressure phase behavior of biomolecules is of biotechnological interest (e.g., high pressure food processing). We demonstrate that temperature and pressure have noncongruent effects on the structural and phase behavior. By using the pressure-jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was also investigated. The time constants for completion of the transitions depend on the direction of the transition, the symmetry and topology of the structures involved, and also on the pressure-jump amplitude. In addition, the effect of incorporating ions, steroids and polypeptides into bilayers on the temperature- and pressure-dependent phase behavior of the lipid systems is discussed.