Two-component Membranes Research Articles

A combination of proteomics, genetics, and physiology provides insights into the acid-tolerance phenotype of Pseudomonas pergaminensis F77

Acid tolerance is crucial for the effective and persistent mineral weathering by acid-producing bacteria. Here, the molecular basis of the acid tolerance of mineral-weathering Pseudomonas pergaminensis F77 was identified using proteomics analysis of the strain under acid stress. Then, the acid tolerance of strain F77 and its mutants with deletion of the acid tolerance-related genes orf03767, mcp, resR, nueR, yegD, and fxsA, which are involved in the two-component systems, DNA repair, nucleotide binding, and membrane parts, were compared. Finally, the acid tolerance-related physiological mechanisms of strain F77 and its mutants F77ΔnueR and F77ΔresR under acidic conditions were characterized. The significantly upregulated proteins in the acid-adapted and acid-challenged strain F77 included the proteins involved in metabolic pathways associated with ATPase, membrane components, organic acid transmembrane transporters, response to stimulus, nucleotide binding, ABC transporters, and two-component systems. The cell numbers decreased by 24–100% at pH ≤ 4.50, while the membrane fluidity increased by 22–61% at pH ≤ 5.50 for the mutants F77ΔnueR and F77ΔresR, compared with that of strain F77. The intracellular H+-ATPase activities decreased by 29–33% for the mutant F77ΔnueR at pH ≤ 4.50% and 33–79% for the mutant F77ΔresR at all tested pHs (pH ≤ 7.00); meanwhile, the ratios of intracellular NAD+/NADH decreased by 71–91% for the mutant F77ΔresR at all tested pHs (pH ≤ 7.00), compared with that of strain F77. Furthermore, the intracellular putrescine concentrations were reduced by 40–70% for the mutant F77ΔresR at all tested pHs (pH ≤ 7.00) compared with that of strain F77. Our findings suggested that multiple proteins and metabolic pathways were associated with bacterial acid tolerance and revealed that nueR and resR were involved in acid tolerance based on their modulation of multiple acid tolerance-related physiological functions in strain F77.

Controlling the shape and topology of two-component colloidal membranes

Changes in the geometry and topology of self-assembled membranes underlie diverse processes across cellular biology and engineering. Similar to lipid bilayers, monolayer colloidal membranes have in-plane fluid-like dynamics and out-of-plane bending elasticity. Their open edges and micrometer-length scale provide a tractable system to study the equilibrium energetics and dynamic pathways of membrane assembly and reconfiguration. Here, we find that doping colloidal membranes with short miscible rods transforms disk-shaped membranes into saddle-shaped surfaces with complex edge structures. The saddle-shaped membranes are well approximated by Enneper's minimal surfaces. Theoretical modeling demonstrates that their formation is driven by increasing the positive Gaussian modulus, which in turn, is controlled by the fraction of short rods. Further coalescence of saddle-shaped surfaces leads to diverse topologically distinct structures, including shapes similar to catenoids, trinoids, four-noids, and higher-order structures. At long timescales, we observe the formation of a system-spanning, sponge-like phase. The unique features of colloidal membranes reveal the topological transformations that accompany coalescence pathways in real time. We enhance the functionality of these membranes by making their shape responsive to external stimuli. Our results demonstrate a pathway toward control of thin elastic sheets' shape and topology-a pathway driven by the emergent elasticity induced by compositional heterogeneity.

Coupling Bulk Phase Separation of Disordered Proteins to Membrane Domain Formation in Molecular Simulations on a Bespoke Compute Fabric.

Phospholipid membranes surround the cell and its internal organelles, and their multicomponent nature allows the formation of domains that are important in cellular signalling, the immune system, and bacterial infection. Cytoplasmic compartments are also created by the phase separation of intrinsically disordered proteins into biomolecular condensates. The ubiquity of lipid membranes and protein condensates raises the question of how three-dimensional droplets might interact with two-dimensional domains, and whether this coupling has physiological or pathological importance. Here, we explore the equilibrium morphologies of a dilute phase of a model disordered protein interacting with an ideal-mixing, two-component lipid membrane using coarse-grained molecular simulations. We find that the proteins can wet the membrane with and without domain formation, and form phase separated droplets bound to membrane domains. Results from much larger simulations performed on a novel non-von-Neumann compute architecture called POETS, which greatly accelerates their execution compared to conventional hardware, confirm the observations. Reducing the wall clock time for such simulations requires new architectures and computational techniques. We demonstrate here an inter-disciplinary approach that uses real-world biophysical questions to drive the development of new computing hardware and simulation algorithms.

Bacillus subtilis natto Derivatives Inhibit Enterococcal Biofilm Formation via Restructuring of the Cell Envelope.

Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, the presence of E. faecalis biofilms can make infections with E. faecalis more difficult to eradicate with current antibiotic therapies. Thus, our aim in this study was to investigate the effects of probiotic derivatives against E. faecalis biofilm formation. Bacillus subtilis natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited adherence to Caco-2 cell monolayers, aggregation, and biofilm production without inhibiting the growth of E. faecalis. An apparent decrease in the thickness of E. faecalis biofilms was observed through confocal laser scanning microscopy. In addition, exopolysaccharide synthesis in E. faecalis biofilms was reduced by B. subtilis natto culture fluid treatment. Carbohydrate composition analysis also showed that carbohydrates in the E. faecalis cell envelope were restructured. Furthermore, transcriptome sequencing revealed that the culture fluid of B. subtilis natto downregulated the transcription of genes involved in the WalK/WalR two-component system, peptidoglycan biosynthesis and membrane glycolipid biosynthesis, which are all crucial for E. faecalis cell envelope synthesis and biofilm formation. Collectively, our work shows that some derivatives present in the culture fluid of B. subtilis natto may be useful for controlling E. faecalis biofilms.

Response of heterotrophic nitrification-aerobic denitrification bacterium Pseudomonas aeruginosa P-1 to Cd2+ and Pb2+ on ammonium removal performance, physiology, and transcriptome analysis

We investigated the response of Pseudomonas aeruginosa P-1, especially when performing heterotrophic nitrification-aerobic denitrification, to Cd2+ and Pb2+ stress. Ammonium removal performance, electron transport system activity (ETSA), and cell morphology were evaluated. The results showed that Pseudomonas aeruginosa P-1 could efficiently remove ammonium at concentrations of 50–200 mg L−1 of Cd2+ and Pb2+. The ETSA maintained a high value at 100 mg L−1 of Cd2+ and Pb2+, and it was less inhibited by Pb2+ than by Cd2+. The results of scanning and transmission electron microscopy showed that most cells had relatively intact surface at 100 mg L−1 of Cd2+ or Pb2+. RNA-Seq transcriptome analysis was employed to identify the mechanisms and key genes involved in the response of P. aeruginosa P-1 to Cd2+ and Pb2+ stress. The results showed that these differentially expressed genes involved in two-component systems and membrane transporters were upregulated. Therefore, P. aeruginosa P-1 may respond to Cd2+ and Pb2+ stress by regulating signal transduction to enable adaption to environmental changes and by varying membrane transporters of the cell membrane to control metal ion access. These findings are of importance in understanding the response mechanism of heterotrophic nitrification-aerobic denitrification bacteria to heavy metals and the potential application of these strains in practical wastewater treatment.

Effect of Selected Anionic and Cationic Drugs Affecting the Central Nervous System on Electrical Properties of Phosphatidylcholine Liposomes: Experiment and Theory.

Interactions between phospholipid membranes and selected drugs affecting the central nervous system (CNS) were investigated. Small, unilamellar liposomes were used as biomimetic cell membrane models. Microelectrophoretic experiments on two-component liposomes were performed using the electrophoretic light scattering technique (ELS). The effect of both positively (perphenazine, PF) and negatively (barbituric acid, BA) charged drugs on zwitterionic L-α-phosphatidylcholine (PC) membranes were analyzed. Experimental membrane surface charge density (δ) data were determined as a function of pH. Quantitative descriptions of the adsorption equilibria formed due to the binding of solution ions to analyzed two-component membranes are presented. Binding constants of the solution ions with perphenazine and barbituric acid-modified membranes were determined. The results of our research show that both charged drugs change surface charge density values of phosphatidylcholine membranes. It can be concluded that perphenazine and barbituric acid are located near the membrane surface, interacting electrostatically with phosphatidylcholine polar heads.

Coarse-grained implicit solvent lipid force field with a compatible resolution to the Cα protein representation.

Biological membranes have been prominent targets for coarse-grained (CG) molecular dynamics simulations. While minimal CG lipid models with three beads per lipid and quantitative CG lipid models with >10 beads per lipid have been well studied, in between them, CG lipid models with a compatible resolution to residue-level CG protein models are much less developed. Here, we extended a previously developed three-bead lipid model into a five-bead model and parameterized it for two phospholipids, POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine). The developed model, iSoLF, reproduced the area per lipid, hydrophobic thickness, and phase behaviors of the target phospholipid bilayer membranes at the physiological temperature. The model POPC and DPPC membranes were in liquid and gel phases, respectively, in accordance with experiments. We further examined the spontaneous formation of a membrane bilayer, the temperature dependence of physical properties, the vesicle dynamics, and the POPC/DPPC two-component membrane dynamics of the CG lipid model, showing some promise. Once combined with standard Cα protein models, the iSoLF model will be a powerful tool to simulate large biological membrane systems made of lipids and proteins.

Molecular dynamics simulations of mechanical stress on oxidized membranes

Biomembranes are under constant attack of free radicals that may lead to lipid oxidation in conditions of oxidative stress. The products generated during lipid oxidation are responsible for structural and dynamical changes which may jeopardize the membrane function. For instance, the local rearrangements of oxidized lipid molecules may induce membrane rupture. In this study, we investigated the effects of mechanical stress on oxidized phospholipid bilayers (PLBs). Model bilayers were stretched until pore formation (or poration) using non-equilibrium molecular dynamics simulations. We studied single-component homogeneous membranes composed of lipid oxidation products, as well as two-component heterogeneous membranes with coexisting native and oxidized domains. In homogeneous membranes, the oxidation products with —OH and —OOH groups reduced the areal strain required for pore formation, whereas the oxidation product with O group behaved similarly to the native membrane. In heterogeneous membranes composed of oxidized and non-oxidized domains, we tested the hypothesis according to which poration may be facilitated at the domain interface region. However, results were inconclusive due to their large statistical variance and sensitivity to simulation setup parameters. We pointed out important technical issues that need to be considered in future simulations of mechanically-induced poration of heterogeneous membranes. This research is of interest for photodynamic therapy and plasma medicine, because ruptured and intact plasma membranes are experimentally considered hallmarks of necrotic and apoptotic cell death.

Myomaker and Myomerger: It Takes Two to Make One

Muscle-forming myotubes are formed from the cell-cell fusion of myoblasts. In this issue of Developmental Cell, Leikina etal. (2018) provide compelling evidence that myoblast fusion is dependent on a two-component membrane fusion complex of Myomaker and Myomerger, which function in hemifusion and pore formation activity, respectively.

Sulfonated Phenylene/Quinquephenylene/Perfluoroalkylene Terpolymers as Proton Exchange Membranes for Fuel Cells

A novel series of terpolymers (SQF) containing sulfophenylene, quinquephenylene, and perfluoroalkylene groups in the polymer main chain were designed and synthesized as proton exchange membranes for fuel cells. The terpolymers with high molecular weight (Mw = 179–207 kDa, Mn = 41–50 kDa) and different ion exchange capacity (IEC) values (1.70, 2.56, and 3.34 mequiv g–1) gave flexible self-standing membranes by solution casting. Compared to the two-component (sulfophenylene and quinquephenylene segments) copolymer membranes, the incorporation of the third component, perfluoroalkylene groups, resulted in better water utilization for the proton conduction, while it did not alter the other properties such as gas permeability and mechanical strength. The selected membrane (SQF-3 with IEC = 2.56 mequiv g–1) exhibited high fuel cell performance under high- and low-humidity conditions with maximum power density reaching 0.97 W cm–2 at 100% RH (relative humidity) and 0.82 W cm–2 at 30% RH, respectively, at a curren...

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

Finsler Geometry Modeling of an Orientation-Asymmetric Surface Model for Membranes

In this paper, a triangulated surface model is studied in the context of Finsler geometry (FG) modeling. This FG model is an extended version of a recently reported model for two-component membranes, and it is asymmetric under surface inversion. We show that the definition of the model is independent of how the Finsler length of a bond is defined. This leads us to understand that the canonical (or Euclidean) surface model is obtained from the FG model such that it is uniquely determined as a trivial model from the viewpoint of well definedness.

Cardiotoxins: Functional Role of Local Conformational Changes.

Cardiotoxins (CTs) from snake venoms are a family of homologous highly basic proteins that have extended hydrophobic patterns on their molecular surfaces. CTs are folded into three β-structured loops stabilized by four disulfide bridges. Being well-structured in aqueous solution, most of these proteins are membrane-active, although the exact molecular mechanisms of CT-induced cell damage are still poorly understood. To elucidate the structure-function relationships in CTs, a detailed knowledge of their spatial organization and local conformational dynamics is required. Protein domain motions can be either derived from a set of experimental structures or generated via molecular dynamics (MD). At the same time, traditional clustering algorithms in the Cartesian coordinate space often fail to properly take into account the local large-scale dihedral angle transitions that occur in MD simulations. This is because such perturbations are usually offset by changes in the neighboring dihedrals, thus preserving the overall protein fold. States with a "locally perturbed" backbone were found in experimental 3D models of some globular proteins and have been shown to be functionally meaningful. In this work, the possibility of large-scale dihedral angle transitions in the course of long-term MD in explicit water was explored for three CTs with different membrane activities: CT 1, 2 (Naja oxiana) and CT A3 (Naja atra). Analysis of the MD-derived distributions of backbone torsion angles revealed several important common and specific features in the structural/dynamic behavior of these proteins. First, large-amplitude transitions were detected in some residues located in the functionally important loop I region. The K5/L6 pair of residues was found to induce a perturbation of the hydrophobic patterns on the molecular surface of CTs-reversible breaking of a large nonpolar zone ("bottom") into two smaller ones and their subsequent association. Second, the characteristic sizes of these patterns perfectly coincided with the dimensions of the nonpolar zones on the surfaces of model two-component (zwitterionic/anionic) membranes. Taken together with experimental data on the CT-induced leakage of fluorescent dye from such membranes, these results allowed us to formulate a two-stage mechanism of CT-membrane binding. The principal finding of this study is that even local conformational dynamics of CTs can seriously affect their functional activity via a tuning of the membrane binding site - specific "hot spots" (like the K5/L6 pair) in the protein structure.

Phase Diagrams and Ordering in Charged Membranes: Binary Mixtures of Charged and Neutral Lipids.

We propose a model describing the phase behavior of two-component membranes consisting of binary mixtures of electrically charged and neutral lipids. We take into account the structural phase transition (main-transition) of the hydrocarbon chains, and investigate the interplay between this phase transition and the lateral phase separation. The presence of charged lipids significantly affects the phase behavior of the multicomponent membrane. Due to the conservation of lipid molecular volume, the main-transition temperature of charged lipids is lower than that of neutral ones. Furthermore, as compared with binary mixtures of neutral lipids, the membrane phase separation in binary mixtures of charged lipids is suppressed, in accord with recent experiments. We distinguish between two types of charged membranes: mixtures of charged saturated lipid/neutral unsaturated lipid and a second case of mixtures of neutral saturated lipid/charged unsaturated lipid. The corresponding phase behavior is calculated and shown to be very different. Finally, we discuss the effect of added salt on the phase separation and the temperature dependence of the lipid molecular area.

Sulfonated poly(ether-ether-ketone) and Nafion composite membrane with aluminium oxide additive for fuel cell applications

The changes of physical properties and proton conductivity due to combining two polymers with inorganic compound are studied. Sulfonated poly(ether-ether-ketone) and Nafion were mixed in proportion 9 to 1; the amount of aluminium oxide nanopowder was 2 wt % counted to polymer part. Also, two-component membranes were prepared for comparison. Resulting membranes were tested for water uptake and impedance spectra were measured at low relative humidity and 70°C temperature. The highest conductivity measured in this work was obtained for ternary membrane with the value of 7.58 mS/cm.

Equilibrium microphase separation in the two-leaflet model of lipid membranes.

Because of the coupling between local lipid composition and the thickness of the membrane, microphase separation in two-component lipid membranes can take place; such effects may underlie the formation of equilibrium nanoscale rafts. Using a kinetic description, this phenomenon is analytically and numerically investigated. The phase diagram is constructed through the stability analysis for linearized kinetic equations, and conditions for microphase separation are discussed. Simulations of the full kinetic model reveal the development of equilibrium membrane nanostructures with various morphologies from the initial uniform state.

Dynamics of two-component membranes surrounded by viscoelastic media

We discuss the dynamics of two-component fluid membranes which are surrounded by viscoelastic media. We assume that membrane-embedded proteins can diffuse laterally and induce a local membrane curvature. The mean squared displacement of a tagged membrane segment is obtained as a generalized Einstein relation. When the elasticity of the surrounding media obeys a power-law behavior in frequency, an anomalous diffusion of the membrane segment is predicted. We also consider the situation where the proteins generate active non-equilibrium forces. The generalized Einstein relation is further modified by an effective temperature that depends on the force dipole energy. The obtained generalized Einstein relations are useful for membrane microrheology experiments.

Probing the importance of lipid diversity in cell membranes via molecular simulation

Lipid membranes in prokaryotes and eukaryotes have a wide array of lipids that are necessary for proper membrane structure and function. In this paper, an introduction to lipid diversity in biology and a mini-review on how molecular simulations have been used to model biological membranes (primarily limited to one to three lipid types in most simulation-based models) is provided, which motivates the use of all-atom molecular dynamics (MD) simulations to study the effect of lipid diversity on properties of realistic membrane models of prokaryotes and eukaryotes. As an example, cytoplasmic membrane models of Escherichia coli were developed at different stages of the colony growth cycle (early-log, mid-log, stationary and overnight). The main difference between lipid compositions at each stage was the concentration of a cyclopropane-containing moiety on the sn-2 lipid acyl chain (cyC17:0). Triplicate MD simulations for each stage were run for 300ns to study the influence of lipid diversity on the surface area per lipid, area compressibility modulus, deuterium order parameters, and electron density profiles. The overnight stage (also known as the death stage) had the highest average surface area per lipid, highest rigidity, and lowest bilayer thickness compare to other stages of E. coli cytoplasmic membrane. Although bilayer thickness did depend on the growth stage, the changes between these were small suggesting that the hydrophobic core of transmembrane proteins fit well with the membrane in all growth stages. Although it is still common practise in MD simulations of membrane proteins to use simple one- or two-component membranes, it can be important to use diverse lipid model membranes when membrane protein structure and function are influenced by changes in lipid membrane composition.

Phase transitions and membrane stiffness in a class of asymmetric heterogeneous fluid membranes

.We propose a minimal model for miscibility phase transitions (MPTs) in a class of asymmetric two-component heterogeneous fluid membranes at equilibrium that generically display both first and second order MPTs, controlled by the interplay of asymmetry and heterogeneity. In the vicinity of the MPTs, the membrane fluctuations are generally enhanced. However, the degree of enhancement is found to depend sensitively on the asymmetry-heterogeneity coupling. We argue that experimental measurements of the membrane fluctuations at the MPTs should provide physical information about the forms of the asymmetry-heterogeneity couplings.

Vesiculation of healthy and defective red blood cells

Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.