Flat Bilayer Research Articles

Increased Binding of Calcium Ions at Positively Curved Phospholipid Membranes.

Calcium ion is the ubiquitous messenger in cells and plays a key role in neuronal signaling and fusion of synaptic vesicles. These vesicles are typically ∼20-50 nm in diameter, and thus their interaction with calcium ions cannot be modeled faithfully with a conventional flat membrane bilayer setup. Within our newly developed molecular dynamics simulations setup, we characterize here interactions of the calcium ion with curved membrane interfaces with atomistic detail. The present molecular dynamics simulations together with time-dependent fluorescence shift experiments suggest that the mode and strength of interaction of calcium ion with a phospholipid bilayer depends on its curvature. Potential of mean force calculations demonstrate that the binding of calcium ion to the positively curved side of the bilayer is significantly stronger compared with that to a flat membrane.

Dual-responsive, shape-switching bilayers enabled by liquid crystal elastomers

Materials that change shape are attractive candidates to replace traditional actuators for applications with power or size restrictions. In this work, we design a polymeric bilayer that changes shape in response to both heat and water by the incorporation of a water-responsive hydrophilic polymer with a heat-responsive liquid crystal elastomer. The distinct shape changes based on stimulus are controlled by the molecular order, and consequently the anisotropic modulus, of a liquid crystal elastomer. In response to water, the hydrophilic polymer layer expands, bending the bilayer along the path dictated by the anisotropic modulus of the liquid crystal elastomer layer, which is approximately 5 times higher along the molecular orientation than in perpendicular directions. We demonstrate that by varying the direction of this stiffer axis in LCE films, helical pitch of the swollen bilayer can be controlled from 0.1 to 20 mm. By spatially patterning the stiffer axis with a resolution of 900 μm2, we demonstrate bilayers that fold and bend based on the pattern within the LCE. In response to heat, the liquid crystal elastomer contracts along the direction of molecular order, and when this actuation is constrained by the hydrophilic polymer, this contraction results in a 3D shape that is distinct from the shape seen in water. Furthermore, by using the vitrification of the dry hydrophilic polymer this 3D shape can be retained in the bilayer after cooling. By utilizing sequential exposure to heat and water, we can drive the initially flat bilayer to reversibly shift between 3D shapes.

Molecular transport through large-diameter DNA nanopores.

DNA-based nanopores are synthetic biomolecular membrane pores, whose geometry and chemical functionality can be tuned using the tools of DNA nanotechnology, making them promising molecular devices for applications in single-molecule biosensing and synthetic biology. Here we introduce a large DNA membrane channel with an ≈4 nm diameter pore, which has stable electrical properties and spontaneously inserts into flat lipid bilayer membranes. Membrane incorporation is facilitated by a large number of hydrophobic functionalizations or, alternatively, streptavidin linkages between biotinylated channels and lipids. The channel displays an Ohmic conductance of ≈3 nS, consistent with its size, and allows electrically driven translocation of single-stranded and double-stranded DNA analytes. Using confocal microscopy and a dye influx assay, we demonstrate the spontaneous formation of membrane pores in giant unilamellar vesicles. Pores can be created both in an outside-in and an inside-out configuration.

3D-Membrane Stacks on Supported Membranes Composed of Diatom Lipids Induced by Long-Chain Polyamines.

Long-chain polyamines (LCPAs) are intimately involved in the biomineralization process of diatoms taking place in silica deposition vesicles being acidic compartments surrounded by a lipid bilayer. Here, we addressed the question whether and how LCPAs interact with lipid membranes composed of glycerophospholipids and glyceroglycolipids mimicking the membranes of diatoms and higher plants. Solid supported lipid bilayers and monolayers containing the three major components that are unique in diatoms and higher plants, i.e., monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), were prepared by spreading small unilamellar vesicles. The integrity of the membranes was investigated by fluorescence microscopy and atomic force microscopy showing continuous flat bilayers and monolayers with small protrusions on top of the membrane. The addition of a synthetic polyamine composed of 13 amine groups separated by a propyl spacer (C3N13) results in flat but three-dimensional membrane stacks within minutes. The membrane stacks are connected with the underlying membrane as verified by fluorescence recovery after photobleaching experiments. Membrane stack formation was found to be independent of the lipid composition; i.e., neither glyceroglycolipids nor negatively charged lipids were required. However, the formation process was strongly dependent on the chain length of the polyamine. Whereas short polyamines such as the naturally occurring spermidine, spermine, and the synthetic polyamines C3N4 and C3N5 do not induce stack formation, those containing seven and more amine groups (C3N7, C3N13, and C3N18) do form membrane stacks. The observed stack formation might have implications for the stability and expansion of the silica deposition vesicle during valve and girdle band formation in diatoms.

Effect of Solvent Dielectric Constant on the Formation of Large Flat Bilayer Stacks in a Lecithin/Hexadecanol Hydrogel.

We investigated the effect of dielectric properties of the aqueous medium on the novel type of hydrogel composed of a crude lecithin mixture (PC70) and hexadecanol (HD), in which charged sheet-like bilayers are kept far apart due to interbilayer repulsive interaction. We used dipropylene glycol (DPG) as a modifier of the dielectric properties and examined its effect on the hydrogel by synchrotron X-ray diffraction, differential scanning calorimetry (DSC), polarized optical microscopy, and freeze-fracture electron microscopy. We found that at a DPG weight fraction in the aqueous medium WDPG ≈ 0.4, the bilayer organization is transformed into unusually large flat bilayer stacks with a regular lamellar spacing of 6.25 nm and consequently disintegration of the hydrogel takes place. Semiquantitative calculation of the interbilayer interaction energy based on the Deyaguin-Landau-Verwey-Overbeek (DLVO) theory suggested that the reduction of the aqueous medium dielectric constant ε by DPG may lower the energy barrier preventing flat bilayers from coming closer together. We inferred that the size of the bilayer sheet increases because the reduction of ε promotes protonation of acidic lipids that work as edge-capping molecules.

Quantum Phase Transition in Germanene and Stanene Bilayer: From Normal Metal to Topological Insulator.

Two-dimensional (2D) topological insulators (TIs) that exhibit quantum spin Hall effects are a new class of materials with conducting edge and insulating bulk. The conducting edge bands are spin-polarized, free of back scattering, and protected by time-reversal symmetry with potential for high-efficiency applications in spintronics. On the basis of first-principles calculations, we show that under external pressure recently synthesized stanene and germanene buckled bilayers can automatically convert into a new dynamically stable phase with flat honeycomb meshes. In contrast with the active surfaces of buckled bilayer of stanene or germanene, the above new phase is chemically inert. Furthermore, we demonstrate that these flat bilayers are 2D TIs with sizable topologically nontrivial band gaps of ∼0.1 eV, which makes them viable for room-temperature applications. Our results suggest some new design principles for searching stable large-gap 2D TIs.

Freezing Transitions of Nanoconfined Coarse-Grained Water Show Subtle Dependence on Confining Environment.

The solid-to-liquid phase transition in water nanofilms confined between plates, with varying separations and water-plate interactions ranging from strongly hydrophobic to strongly hydrophilic, was simulated using a coarse-grained monatomic water model (mW) and the generalized replica exchange method (gREM). Extensive gREM simulations combined with the statistical temperature weighted histogram analysis method (ST-WHAM) provide a detailed description of the thermodynamic properties intrinsic to the phase transition, including the transition temperature, isobaric heat capacity, phase change enthalpy, entropy, and their dependence on the interplate distance and the plate-water interaction. The ice structure of water nanofilms was characterized at various conditions using the transverse density profile and the distribution of angles formed by hydrogen-bonded neighboring molecules. Flat bilayer ice was observed to be the dominant solid phase at close interplate distance, while puckered bilayer ice, similar to a slab of ice Ih, is the predominant structure at larger interplates. Stable puckered bilayer ice, previously observed to have a low melting point, is observed to have enhanced stability with high melting temperature when confined between hydrophilic plates. These results demonstrate the strong dependence of phase stability and coexistence in nanoconfined systems on the geometry and physical properties of the confining environment.

Membrane-bound MinDE complex acts as a toggle switch that drives Min oscillation coupled to cytoplasmic depletion of MinD

The Escherichia coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to prevent divisions at the cell poles. Reconstituting the Min system on a lipid bilayer has contributed to elucidating the oscillatory mechanism. However, previous in vitro patterns were attained with protein densities on the bilayer far in excess of those in vivo and failed to recapitulate the standing wave oscillations observed in vivo. Here we studied Min protein patterning at limiting MinD concentrations reflecting the in vivo conditions. We identified "burst" patterns--radially expanding and imploding binding zones of MinD, accompanied by a peripheral ring of MinE. Bursts share several features with the in vivo dynamics of the Min system including standing wave oscillations. Our data support a patterning mechanism whereby the MinD-to-MinE ratio on the membrane acts as a toggle switch: recruiting and stabilizing MinD on the membrane when the ratio is high and releasing MinD from the membrane when the ratio is low. Coupling this toggle switch behavior with MinD depletion from the cytoplasm drives a self-organized standing wave oscillator.

Fusion Between v-SNARE Nanodiscs and “Flipped” t-SNARE Cells: Control of Fusion Pore Nucleation and Lifetimes by SNARE Protein Transmembrane Domains

A key step during neurotransmitter or hormone release (exocytosis) is the formation of a nanometer-sized fusion pore that connects the plasma membrane to the synaptic or secretory vesicle. The pore can flicker open and closed repeatedly before dilating or resealing irreversibly. Pore dynamics affect vesicle recycling, amount and kinetics of released cargo, and activation of downstream events. The fusion pore is clearly a key intermediate; however, factors regulating its dynamics are poorly understood, in large part due to a lack of biochemically defined single-pore assays.We have developed a novel assay that probes single fusion pores formed between ∼16 nm flat lipid bilayer nanodiscs (NDs) reconstituted with neuronal/exocytotic vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) proteins and cells engineered to express cognate “flipped” target membrane t-SNAREs on their surfaces, with the SNARE domain facing the extracellular medium. Conductance through single, voltage-clamped fusion pores directly reports sub-millisecond pore dynamics. Pore currents fluctuated, briefly returned to baseline multiple times, and disappeared ∼6 s after initial opening, as if the fusion pore fluctuated in size, flickered, and resealed. We found that putative interactions between v- and t-SNARE transmembrane domains (TMDs) promote, but are not essential for pore nucleation. Surprisingly, the same modifications that reduced pore nucleation rates dramatically prolonged pore lifetimes. Our results indicate that rod-shaped, post-fusion cis-SNARE complexes that poorly fit the highly curved fusion site rapidly vacate the pore region, leaving pore properties to be determined by lipids. In contrast, mutants deficient in TMD-zippering prevented pore resealing for >60 s, possibly due to their post-fusion Y-shape. Thus, post-fusion geometry of the proteins may determine pore stability, analogous to the well-known effects of lipid geometry on highly curved fusion intermediates.

SNARE-Mediated Transient Fusion of Liposomes to Supported Bilayers Probed by Two-Color pTIRFM

In membrane fusion opening of a fusion pore establishes the first connection between two compartments. During neurotransmitter or hormone release via exocytosis, fusion pores can transiently open and close repeatedly (flicker) before resealing (‘kiss-and-run’) or dilating irreversibly (full fusion). Pore dynamics regulate the amount and size of released cargo and determine the mode of vesicle recycling, yet what governs pore dynamics is poorly understood. We established an assay to monitor membrane fusion by polarized total internal reflection (pTIRF) microscopy with single molecule sensitivity and ∼15 ms time resolution in a biochemically well-defined system. In our assay, fusion of small unilamellar vesicles containing v-SNARE proteins (v-SUVs) fuse to a flat bilayer reconstituted with cognate t-SNAREs, supported on a soft polymer cushion (t-SBL). Exploiting the signal enhancement when a label transfers from the SUV to the SBL membrane in pTIRF, we could monitor the release kinetics of vesicular lipid labels into the SBL upon fusion. We found release times were much longer than would be expected for permanently open pores. Our sensitivity to single fluorescent lipids allowed us to calculate the lipid diffusivity and SUV size for each fusion event. This was used in a model we developed assuming the retardation was due to flickering pores, to estimate pore openness P0, the fraction of time a pore remains open during fusion. Increasing cholesterol levels increased P0. However, retardation of lipid release could also be due to steric hindrance of lipid diffusion through the walls of the fusion pore by SNARE proteins. To exclude this, we encapsulated soluble markers into the SUVs and simultaneously monitored lipid and contents release. Release of both lipid and soluble cargo started simultaneously. Interestingly, many pores resealed after partially releasing their contents.

Reverse osmosis membranes based on a supported lipid bilayer with gramicidin A water channels

This study presents the fabrication of a biomimetic reverse osmosis (RO) membrane from a supported lipid bilayer (SLB) and gramicidin A (GA) onto polymeric substrates via electrostatic interaction and by applying hydraulic pressure. GA, a cation-selective ionophore, has high permeability for water molecules and monovalent cations in a lipid bilayer, and was therefore used as a water channel molecule. Cationic liposomes containing GA were electrostatically adsorbed onto an anionic polymeric nanofiltration membrane. The adsorbed liposomes were ruptured by applying hydraulic pressure and transformed into flat lipid bilayers. SLB formation was confirmed by salt rejection measurement and fluorescence recovery after photobleaching. The defect-free SLB was formed on the polymeric nanofiltration membrane by applying over 0.15MPa pressure. Phosphorus quantification showed that the formed SLB was not composed of a single lipid bilayer but approximately six lipid bilayers. GA incorporation into the SLB and control of the GA conformation on the SLB effectively improved water permeability. Moreover, the GA-incorporated SLB showed high NaCl rejection (>97%). Monovalent cations, Na+ ions, were rejected to maintain electric neutrality. The formed SLB showed hydraulic pressure resistance and high salt rejection at adequate levels for RO membranes.

Synaptobrevin Transmembrane Domain Influences Exocytosis by Perturbing Vesicle Membrane Curvature

Membrane fusion requires that nearly flat lipid bilayers deform into shapes with very high curvature. This makes membrane bending a critical force in determining fusion mechanisms. A lipid bilayer will bend spontaneously when material is distributed asymmetrically between its two monolayers, and its spontaneous curvature (C0) will influence the stability of curved fusion intermediates. Prior work on Ca2+-triggered exocytosis revealed that fusion pore lifetime (τ) varies with vesicle content (Q), and showed that this relation reflects membrane bending energetics. Lipids that alter C0 change the dependence of τ on Q. These results suggested that the greater stability of an initial exocytotic fusion pore associated with larger vesicles reflects the need to bend more membrane during fusion pore dilation. In this study, we explored the possibility of manipulating C0 by mutating the transmembrane domain (TMD) of the vesicle membrane protein synaptobrevin 2 (syb2). Amperometric measurements of exocytosis in mouse chromaffin cells revealed that syb2 TMD mutations altered the relation between τ and Q. The effects of these mutations showed a striking periodicity, changing sign as the structural perturbation moved through the inner and outer leaflets. Some glycine and charge mutations also influenced the dependence of τ on Q in a manner consistent with expected changes in C0. These results suggest that side chains in the syb2 TMD influence the kinetics of exocytosis by perturbing the packing of the surrounding lipids. The present results support the view that membrane bending occurs during fusion pore expansion rather than during fusion pore formation. This supports the view of an initial fusion pore through two relatively flat membranes formed by protein.

Small-Angle X-Ray Scattering of the Cholesterol Incorporation into Human ApoA1-POPC Discoidal Particles

Structural and functional aspects of high-density lipoproteins have been studied for over half a century. Due to the plasticity of this highly complex system, new aspects continue to be discovered. Here, we present a structural study of the human Apolipoprotein A1 (ApoA1) and investigate the role of its N-terminal domain, the so-called globular domain of ApoA1, in discoidal complexes with phospholipids and increasing amounts of cholesterol. Using a combination of solution-based small-angle x-ray scattering (SAXS) and molecular constrained data modeling, we show that the ApoA1-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-based particles are disk shaped with an elliptical cross section and composed by a central lipid bilayer surrounded by two stabilizing ApoA1 proteins. This structure is very similar to the particles formed in the so-called nanodisc system, which is based on N-terminal truncated ApoA1 protein. Although it is commonly agreed that the nanodisc is plain disk shaped, several more advanced structures have been proposed for the full-length ApoA1 in combination with POPC and cholesterol. This prompted us to make a detailed comparative study of the ApoA1 and nanodisc systems upon cholesterol uptake. Based on the presented SAXS analysis it is found that the N-terminal domains of ApoA1-POPC-cholesterol particles are not globular but instead an integrated part of the protein belt stabilizing the particles. Upon incorporation of increasing amounts of cholesterol, the presence of the N-terminal domain allows the bilayer thickness to increase while maintaining an overall flat bilayer structure. This is contrasted by the energetically more strained and less favorable lens shape required to fit the SAXS data from the N-terminal truncated nanodisc system upon cholesterol incorporation. This suggests that the N-terminal domain of ApoA1 actively participates in the stabilization of the ApoA1-POPC-cholesterol discoidal particle and allows for a more optimal lipid packing upon cholesterol uptake.

Molecular dynamics simulations of liquid crystalline phases of dodecyltrimethylammonium chloride

Molecular dynamics simulations of four different phases of a cationic surfactant dodecyltrimethylammonium chloride (DTAC) are presented. It is shown that when the topology of the initial configuration matches that of the equilibrium structure, the required equilibration times of MD simulations are only few nanoseconds. The methods of building initial configurations for simulations of the hexagonal and Ia3d bicontinuous cubic phases are described. The simulation results show that locally, the hydrophilic part of the hexagonal phase has a flat bilayer structure. Analysis of radial distribution functions shows that the properties of the hydrophilic layers of the phases are dominated by ion–ion and ion–water interactions. The dynamic properties of the system are dependent on the curvature of the aggregates, and calculated diffusion coefficients are in agreement with experimental NMR data.

Spatial Regulators for Bacterial Cell Division Self-Organize into an Oscillator on a Flat Bilayer

The E. coli Min system self-organizes into a cell-pole to cell-pole oscillator on the membrane to position cell division at mid-cell. We reconstituted and visualized the Min system on a flat lipid bilayer under protein depletion conditions and identified a pattern we call ‘bursts’ - radially expanding bilayer binding centers of the MinD ATPase that are corralled and disassembled by a ring of the ATPase stimulator, MinE. Bursts share several features with in vivo dynamics such as the ability to oscillate. We propose a patterning mechanism whereby the Min system self-organizes into an oscillator without the spatial confinement of a bacterial cell.

Determining Material Elastic Properties of Arbitrarily-Shaped Membranes using Molecular Dynamics Simulations with Application to the Inverted Hexagonal Phase

Accumulating evidence indicates that diverse physiological processes are influenced by the lipid composition of the membrane and by its material properties. This has notably been shown for the function of diverse proteins and their oligomerization, and processes on larger scales such as membrane reshaping and fusion. Determination of the elastic properties of lipidic membranes is therefore of great importance to our understanding of these processes. Experimental approaches to determine the material properties of lipids remain challenging and usually rely on their study in a relaxed environment or in flat bilayers, although it is widely accepted that cell membranes can be under considerable stress and frustration as well as high local curvature. Whether this impacts the measured properties is a matter of debate so that studying membranes under more realistic conditions is key for our understanding how these material properties impact different physiological processes. In this context, we propose a computational method to determine the elastic properties of lipid assemblies of arbitrarily shaped interfaces and use it to study the impact of the curvature of a membrane on its elastic properties. Specifically, we apply the methodology to mixtures of DOPE (dioleoylphosphatidylethanolamine) lipids and cholesterol in the inverted-hexagonal and lamellar phases and find that the bending rigidity for a particular lipid composition critically depends on the geometry of the lipidic system. This dependence correlates on the molecular level to the changes in lipid chain order imposed by the membrane curvature, implying that these results should pertain to other situations where the membrane is deformed, stressed or frustrated that notably emerge around integral membrane proteins or during membrane remodeling processes such as budding.

Specific adhesion of membranes simultaneously supports dual heterogeneities in lipids and proteins.

Membrane adhesion is a vital component of many biological processes. Heterogeneities in lipid and protein composition are often associated with the adhesion site. These heterogeneities are thought to play functional roles in facilitating signalling. Here we experimentally examine this phenomenon using model membranes made of a mixture of lipids that is near a phase boundary at room temperature. Non-adherent model membranes are in a well-mixed, disordered-fluid lipid phase indicated by homogeneous distribution of a fluorescent dye that is a marker for the fluid-disordered (Ld) phase. We specifically adhere membranes to a flat substrate bilayer using biotin-avidin binding. Adhesion produces two types of coexisting heterogeneities: an ordered lipid phase that excludes binding proteins and the fluorescent membrane dye, and a disordered lipid phase that is enriched in both binding proteins and membrane dye compared with the non-adhered portion of the same membrane. Thus, a single type of adhesion interaction (biotin-avidin binding), in an initially-homogeneous system, simultaneously stabilizes both ordered-phase and disordered-phase heterogeneities that are compositionally distinct from the non-adhered portion of the vesicle. These heterogeneities are long-lived and unchanged upon increased temperature.

Curvature and Lipid Packing Modulate the Elastic Properties of Lipid Assemblies: Comparing HII and Lamellar Phases.

Accumulating evidence indicates that membrane reshaping and fusion processes, as well as regulation of membrane protein function, depend on lipid composition. Although it is widely accepted that cell membranes are under considerable stress and frustration and can be locally highly curved, experimental approaches to determine the material properties of lipids usually rely on their study in a relaxed environment or in flat bilayers. Here, we propose a computational method to determine the elastic properties of lipid assemblies of arbitrarily shaped interfaces and apply it to lipidic mixtures in the inverted hexagonal and lamellar phases. We find that the bending rigidity critically depends on the geometry of the system and correlates with the changes in lipid chain order imposed by the specific environment. Our results are relevant for resolving local lipid properties of deformed, stressed, or frustrated membranes that notably emerge around integral membrane proteins or during different membrane remodeling processes.

Correlating Non-Geminate Recombination with Film Structure: A Comparison of Polythiophene: Fullerene Bilayer and Blend Films.

The morphology of the active layer in polymer:fullerene solar cells is a key parameter in determining their performance. In this study we monitor the charge carrier dynamics in bilayers of poly(3-hexylthiophene) (P3HT) and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) (fabricated by sequential spin coating and vacuum deposition) before and after thermal annealing, and compare these against conventional solution processed bulk heterojunction (BHJ) blend films. Transmission electron microscopy images, supported by field effect mobility data show that while not-annealed P3HT/PC61BM bilayers possess a sharp interface, intermixing proceeds upon annealing. Transient absorption studies indicate that the not-annealed bilayer yields fewer, but longer lived, charge carriers compared to the BHJ. Monte Carlo (MC) simulations further suggest that the difference in non-geminate recombination dynamics observed for the BHJ and bilayer films could be related to the confinement of charge carriers to the interface, with the lower dimensionality for the flat interface bilayer films relative to the intercalated donor-acceptor network BHJ films leading to lower recombination.

Novel Insights into the Mechanistic Routes of Lyotropic Phase Transitions

When mixed with water, biological amphiphiles such as phospholipids can self-assemble to form a variety of lyotropic liquid crystalline structures including 1-D flat lamellar bilayers, 2-D hexagonal phases and 3-D bicontinuous cubic structures1. The role of the lamellar phase in nature is well understood – flat bilayers maintain the fundamental integrity of all living cells. However, non-lamellar phases also play a vital role in vivo. Extended cubic phases have been directly observed in cells and in addition, the repeating pores which form the continuously accessible structure of these bicontinuous phases are very closely structurally related to membrane pores formed during membrane fusion and fission. The equilibrium phase behaviour of pure phospholipids in water and simple model membrane mixtures has been widely studied but their out of equilibrium behaviour remains poorly understood. We have addressed this knowledge gap through pioneering work looking at the kinetics of phase transitions in phospholipid/water systems and phospholipid/protein/water mixtures using the pressure jump relaxation technique in conjunction with high speed, time resolved small angle X-ray diffraction. This presentation will provide an overview of how our bottom-up approach using model membrane systems has enabled us to establish the mechanistic routes and intermediates in a number of phase transition schemes including bicontinuous cubic to lamellar, hexagonal to lamellar and inter-cubic phase transitions.