Asymmetric Model Membranes Research Articles

Electroporation of asymmetric phospholipid membranes.

As plasma membranes of animal cells are known to be asymmetric, the transmembrane lipid asymmetry, being essential for many membranes' properties and functions, should be properly accounted for in model membrane systems. In this paper, we employ atomic-scale molecular dynamics simulations to explore electroporation phenomena in asymmetric model membranes comprised of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid monolayers that mimic the outer and inner leaflets of plasma membranes, respectively. Our findings clearly demonstrate that the molecular mechanism of electroporation in asymmetric phospholipid membranes differs considerably from the picture observed for their single-component symmetric counterparts: The initial stages of electric-field-induced formation of a water-filled pore turn out to be asymmetric and occur mainly on the PC side of the PC/PE membrane. In particular, water molecules penetrate in the membrane interior mostly from the PC side, and the reorientation of lipid head groups, being crucial for stabilizing the hydrophilic pore, also takes place in the PC leaflet. In contrast, the PE lipid head groups do not enter the central region of the membrane until the water pore becomes rather large and partly stabilized by PC head groups. Such behavior implies that the PE leaflet is considerably more robust against an electric field most likely due to interlipid hydrogen bonding. We also show that an electric field induces asymmetric changes in the lateral pressure profile of PC/PE membranes, decreasing the cohesion between lipid molecules predominantly in the PC membrane leaflet. Overall, our simulations provide compelling evidence that the transmembrane lipid asymmetry can be essential for understanding electroporation phenomena in living cells.

Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry

Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes “artificial plasma membrane mimicking” (“PMm”) vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

Transbilayer Coupling of Lipid Dynamics

Lipids are organized in plasma membranes in a bilayer with distinctive outer exoplasmic and inner cytoplasmic leaflets. These leaflets differ greatly in terms of their lipid and attached protein compositions. In addition to this asymmetry, membranes are also heterogeneous with respect to their lateral organization (1,2). Recently, model membranes have been developed that are able to mimic lateral heterogeneity as well as transbilayer asymmetry of their constituent lipids (3,4). In these systems, the exoplasmic mimicking leaflets contain high-chain-melting temperature lipids, low-chain-melting temperature lipids, and cholesterol. These ternary mixtures, by themselves, form phase-separated membranes with microscopic lipid-raft-like liquid-ordered and liquid-disordered domains. It turns out that in asymmetric model membranes, these domains can induce the formation of domains in the opposing leaflet even when they are composed of lipids that, by themselves, wouldn’t separate into different phases. This is important, because it might be one mechanism by which signaling (raft-) components are brought together at the two sides of the plasma membrane. Recently, it has been shown that this process modulates the spatial distribution of the anionic lipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate, which then modulates the binding of C2 domains of the synaptic vesicle-membrane protein synaptotagmin1 (5). Which interactions are responsible for this lipid-mediated transbilayer coupling? A few theoretical models have been published in which acyl-chain interdigitation, midplane surface tension, transbilayer cholesterol dynamics, and electrostatic interactions are discussed as possible mechanisms (6,7). However, these interactions had not been systematically explored previously. In this volume of the Biophysical Journal, Chiantia and London (8) are taking first steps toward a deeper understanding of this phenomenon by reporting results about the influence of acyl-chain length and saturation on the interleaflet coupling in asymmetric bilayers. The authors make use of their recently developed liposome-based asymmetric membrane system (9,10), with which they can determine diffusion coefficients (by fluorescence correlation spectroscopy) and order parameters (from fluorescence lifetime) in the two leaflets of the lipid bilayer independently. Simple lipid compositions were used in both leaflets: the inner leaflet contained only a single species of phosphatidylcholine (PC) and the outer leaflet contained a mixture of sphingomyelin and PC. Stronger coupling, measured by a reduced diffusion coefficient in the inner PC leaflet, was achieved by sphingomyelin lipids with longer acyl chains in the outer leaflet and/or by PC lipids with one saturated and one unsaturated acyl chain in the inner leaflet. Surprisingly, the packing order of the PC-leaflet was not influenced by the more ordered outer leaflet. Even though these results cannot explain the coupling between coexisting lipid phases in asymmetric lipid raft membranes, they are exciting because they emphasize the role of lipid clusters or domains that go beyond those of lipid rafts. Under the described conditions, clusters of slowly diffusing lipids can induce transient domains that are defined by their diffusion dynamics rather than their acyl-chain order. This type of coupling should happen independently of how the inducing lipid cluster was formed in the first place. Possible mechanisms include interactions with integral or peripheral membrane proteins, or the already mentioned phase separation in raftlike lipid mixtures. Chiantia and London present a methodology that, in the future, perhaps in combination with recent developments in super-resolution microscopy (11), will give us a better understanding of the molecular underpinning of lipid-mediated interleaflet coupling. In particular, it will be interesting to see how the inclusion of cholesterol or proteins will affect lipid dynamics and membrane order in model and cell membranes.

Conformational dynamics and membrane interactions of the E. coli outer membrane protein FecA: A molecular dynamics simulation study

The TonB-dependent transporters mediate high-affinity binding and active transport of a variety of substrates across the outer membrane of Escherichia coli. The substrates transported by these proteins are large, scarce nutrients that are unable to gain entry into the cell by passive diffusion across the complex, asymmetric bilayer that constitutes the outer membrane. Experimental studies have identified loop regions that are essential for the correct functioning of these proteins. A number of these loops have been implicated in ligand binding. We report the first simulations of an E. coli outer membrane protein in an asymmetric model membrane that incorporates lipopolysaccharide (LPS) molecules. Comparative simulations of the apo and holo forms of the TonB-dependent transporter FecA in different membrane models enable us to identify the nature of the LPS–protein interactions and determine how these interactions impact upon the conformational dynamics of this protein. In particular, our simulations provide molecular-level insights into the influence of the environment and ligand on the dynamics of the functionally important loops of FecA. In addition, we provide insights into the nature of the protein–ligand interactions and ligand induced conformational change in FecA.

Interfacial acid–base equilibrium and electrostatic potentials of model Langmuir–Blodgett membranes in contact with phosphate buffer

The effect of a cationic Langmuir–Blodgett (LB) multilayer of varying thickness on p K i of the weak acid 4-heptadecyl-7-hydroxy coumarin (HHC) located at the multilayer-phosphate buffer interface is investigated. The amphiphilic fluorescent dye is embedded in a neutral methyl arachidate (MA) monolayer that has been deposited atop of the LB-film during dipping. Such a system is considered as an asymmetric model membrane built up of docosyl ammonium cations and phosphate counter-ions (HPO 4 2−). Its electrostatic properties are compared with those of the previously studied LB-membrane of Cd-arachidate [1] consisting of arachidate anions and Cd 2+ counter-ions. The fluorimetric titration of HHC in the neutral matrix at the multilayer-phosphate buffer interface shows that p K m/w at the membrane–water interface increases with increasing membrane thickness, reaching saturation at about 250 Å. The plateau of p K m/w=6.8 found for this system significantly differs from the plateau value of 10.6, observed for the Cd-arachidate multilayer-phosphate buffer boundary [1], and from p K a/w=8.2 obtained at the air–phosphate buffer interface [2]. The shifts, Δp K i=p K a/w−p K m/w, imply positive membrane–water Gouy–Chapman potentials for all studied thicknesses of docosyl ammonium phosphate (DCAP) multilayer, and negative ψ m/w potentials for all Cd-arachidate membranes. The plateau values, obtained for membranes more then 250 Å thick, are ψ m/w ∞=+81 and −195 mV, respectively. Thus, the membrane–water interface appears charged, in spite of the presence of a neutral MA monolayer at the LB-film-phosphate buffer boundary. The ψ m/w/ d dependencies can be well fitted with the equation ψ m w = ψ s m ∞+ψ m w ∞ cosh κd sinh κd describing the variation of the double layer potentials of asymmetric thin films with film thickness at constant surface charge densities [21a]. The fit of this equation to the experimental data for ψ m/w /d yields the Gouy–Chapman potential at the glass-LB membrane interface, ψ s/m ∞ and the Debye length, κ −1, of the diffuse double layers at the s/m and m/w interfaces. The values obtained show that the cationic membrane has two positively charged surfaces with different (small) charge densities, while the anionic membrane has a positive s/m and a negative m/w interface, both having large charge densities. The values of κ −1 enable estimation of the concentration of the free divalent charges in the membrane, which appear to be of the order of (4–5)×10 22 m −3. The dipole potential at the interface cationic membrane–phosphate buffer is positive and linearly increases with increasing membrane thickness. It results from the different density of DCAP dipoles deposited on the solid substrate at dipping and withdrawal. For anionic membrane the density of Cd-arachidate dipoles in the adjacent monolayers is the same, and only the last MA monolayer contributes a small positive dipole potential that is independent on membrane thickness.