Phase Separation in Atomistic Simulations of Model Membranes.

Understanding the lateral organization in plasma membranes remains an open problem despite a large body of research. Model membranes with coexisting micrometer-size domains are routinely employed as simplified models of plasma membranes. Many molecular dynamics simulations have investigated phase separation in model membranes at the coarse-grained level, but atomistic simulations remain computationally challenging. We simulate DPPC:DOPC and DPPC:DOPC:cholesterol lipid bilayers to investigate phase transitions at temperatures from 310 to 270 K. In this temperature range, the binary mixture forms a liquid phase (Lα) and a coexistence of Lα and either gel or ripple phases. The ternary mixture forms a liquid disordered (Ld) phase and a coexistence of liquid ordered (Lo) and either Ld or gel phases. We quantify the coexisting phases and discuss their properties against the background of experimental results. We observe partial registration of growing domains in both mixtures. We characterize specific cholesterol-cholesterol and cholesterol-phospholipid interaction geometries underlying its increased partitioning and the smoothed phase transition in the ternary mixture compared to the binary mixture. By comparing coexisting domains with homogeneous bilayers of the same composition, we demonstrate how domain coexistence affects their properties. Our simulations provide important insights into the lipid-lipid interactions in model lipid bilayers and improve our understanding of the lateral organization in plasma membranes with higher compositional complexity.

Composition Fluctuations in Lipid Bilayers.

Cell membranes contain multiple lipid and protein components having heterogeneous in-plane (lateral) distribution. Nanoscale rafts are believed to play an important functional role, but their phase state-domains of coexisting phases or composition fluctuations-is unknown. As a step toward understanding lateral organization of cell membranes, we investigate the difference between nanoscale domains of coexisting phases and composition fluctuations in lipid bilayers. We simulate model lipid bilayers with the MARTINI coarse-grained force field on length scales of tens of nanometers and timescales of tens of microseconds. We use a binary and a ternary mixture: a saturated and an unsaturated lipid, or a saturated lipid, an unsaturated lipid, and cholesterol, respectively. In these mixtures, the phase behavior can be tuned from a mixed state to a coexistence of a liquid-crystalline and a gel, or a liquid-ordered and a liquid-disordered phase. Transition from a two-phase to a one-phase state is achieved by raising the temperature and adding a hybrid lipid (with a saturated and an unsaturated chain). We analyze the evolution of bilayer properties along this transition: domains of two phases transform to fluctuations with local ordering and compositional demixing. Nanoscale domains and fluctuations differ in several properties, including interleaflet overlap and boundary length. Hybrid lipids show no enrichment at the boundary, but decrease the difference between the coexisting phases by ordering the disordered phase, which could explain their role in cell membranes.

Computer simulations of lung surfactant.

Lung surfactant lines the gas-exchange interface in the lungs and reduces the surface tension, which is necessary for breathing. Lung surfactant consists mainly of lipids with a small amount of proteins and forms a monolayer at the air-water interface connected to bilayer reservoirs. Lung surfactant function involves transfer of material between the monolayer and bilayers during the breathing cycle. Lipids and proteins are organized laterally in the monolayer; selected species are possibly preferentially transferred to bilayers. The complex 3D structure of lung surfactant and the exact roles of lipid organization and proteins remain important goals for research. We review recent simulation studies on the properties of lipid monolayers, monolayers with phase coexistence, monolayer-bilayer transformations, lipid-protein interactions, and effects of nanoparticles on lung surfactant. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

The mechanism of collapse of heterogeneous lipid monolayers.

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10-25 µs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.

Molecular structure of membrane tethers.

Membrane tethers are nanotubes formed by a lipid bilayer. They play important functional roles in cell biology and provide an experimental window on lipid properties. Tethers have been studied extensively in experiments and described by theoretical models, but their molecular structure remains unknown due to their small diameters and dynamic nature. We used molecular dynamics simulations to obtain molecular-level insight into tether formation. Tethers were pulled from single-component lipid bilayers by application of an external force to a lipid patch along the bilayer normal or by lateral compression of a confined bilayer. Tether development under external force proceeded by viscoelastic protrusion followed by viscous lipid flow. Weak forces below a threshold value produced only a protrusion. Larger forces led to a crossover to tether elongation, which was linear at a constant force. Under lateral compression, tethers formed from undulations of unrestrained bilayer area. We characterized in detail the tether structure and its formation process, and obtained the material properties of the membrane. To our knowledge, these results provide the first molecular view of membrane tethers.

Molecular view of phase coexistence in lipid monolayers.

We used computer simulations to study the effect of phase separation on the properties of lipid monolayers. This is important for understanding the lipid-lipid interactions underlying lateral heterogeneity (rafts) in biological membranes and the role of domains in the regulation of surface tension by lung surfactant. Molecular dynamics simulations with the coarse-grained MARTINI force field were employed to model large length (~80 nm in lateral dimension) and time (tens of microseconds) scales. Lipid mixtures containing saturated and unsaturated lipids and cholesterol were investigated under varying surface tension and temperature. We reproduced compositional lipid demixing and the coexistence of liquid-expanded and liquid-condensed phases as well as liquid-ordered and liquid-disordered phases. Formation of the more ordered phase was induced by lowering the surface tension or temperature. Phase transformations occurred via either nucleation or spinodal decomposition. In nucleation, multiple domains formed initially and subsequently merged. Using cluster analysis combined with Voronoi tessellation, we characterized the partial areas of the lipids in each phase, the phase composition, the boundary length, and the line tension under varying surface tension. We calculated the growth exponents for nucleation and spinodal decomposition using a dynamical scaling hypothesis. At low surface tensions, liquid-ordered domains manifest spontaneous curvature. Lateral diffusion of lipids is significantly slower in the more ordered phase, as expected. The presence of domains increased the monolayer surface viscosity, in particular as a result of domain reorganization under shear.

Lung surfactant protein SP-B promotes formation of bilayer reservoirs from monolayer and lipid transfer between the interface and subphase.

We investigated the possible role of SP-B proteins in the function of lung surfactant. To this end, lipid monolayers at the air/water interface, bilayers in water, and transformations between them in the presence of SP-B were simulated. The proteins attached bilayers to monolayers, providing close proximity of the reservoirs with the interface. In the attached aggregates, SP-B mediated establishment of the lipid-lined connection similar to the hemifusion stalk. Via this connection, a lipid flow was initiated between the monolayer at the interface and the bilayer in water in a surface-tension-dependent manner. On interface expansion, the flow of lipids to the monolayer restored the surface tension to the equilibrium spreading value. SP-B induced formation of bilayer folds from the monolayer at positive surface tensions below the equilibrium. In the absence of proteins, lipid monolayers were stable at these conditions. Fold nucleation was initiated by SP-B from the liquid-expanded monolayer phase by local bending, and the proteins lined the curved perimeter of the growing fold. No effect on the liquid-condensed phase was observed. Covalently linked dimers resulted in faster kinetics for monolayer folding. The simulation results are in line with existing hypotheses on SP-B activity in lung surfactant and explain its molecular mechanism.

The molecular mechanism of lipid monolayer collapse.

Lipid monolayers at an air-water interface can be compressed laterally and reach high surface density. Beyond a certain threshold, they become unstable and collapse. Lipid monolayer collapse plays an important role in the regulation of surface tension at the air-liquid interface in the lungs. Although the structures of lipid aggregates formed upon collapse can be characterized experimentally, the mechanism leading to these structures is not fully understood. We investigate the molecular mechanism of monolayer collapse using molecular dynamics simulations. Upon lateral compression, the collapse begins with buckling of the monolayer, followed by folding of the buckle into a bilayer in the water phase. Folding leads to an increase in the monolayer surface tension, which reaches the equilibrium spreading value. Immediately after their formation, the bilayer folds have a flat semielliptical shape, in agreement with theoretical predictions. The folds undergo further transformation and form either flat circular bilayers or vesicles. The transformation pathway depends on macroscopic parameters of the system: the bending modulus, the line tension at the monolayer-bilayer connection, and the line tension at the bilayer perimeter. These parameters are determined by the system composition and temperature. Coexistence of the monolayer with lipid aggregates is favorable at lower tensions of the monolayer-bilayer connection. Transformation into a vesicle reduces the energy of the fold perimeter and is facilitated for softer bilayers, e.g., those with a higher content of unsaturated lipids, or at higher temperatures.

Direct simulation of protein-mediated vesicle fusion: lung surfactant protein B.

We simulated spontaneous fusion of small unilamellar vesicles mediated by lung surfactant protein B (SP-B) using the MARTINI force field. An SP-B monomer triggers fusion events by anchoring two vesicles and facilitating the formation of a lipid bridge between the proximal leaflets. Once a lipid bridge is formed, fusion proceeds via a previously described stalk - hemifusion diaphragm - pore-opening pathway. In the absence of protein, fusion of vesicles was not observed in either unbiased simulations or upon application of a restraining potential to maintain the vesicles in close proximity. The shape of SP-B appears to enable it to bind to two vesicles at once, forcing their proximity, and to facilitate the initial transfer of lipids to form a high-energy hemifusion intermediate. Our results may provide insight into more general mechanisms of protein-mediated membrane fusion, and a possible role of SP-B in the secretory pathway and transfer of lung surfactant to the gas exchange interface.

Cholesterol Flip-Flop in Heterogeneous Membranes.

Cholesterol is the most abundant molecule in the plasma membrane of mammals. Its distribution across the two membrane leaflets is critical for understanding how cells work. Cholesterol trans-bilayer motion (flip-flop) is a key process influencing its distribution in membranes. Despite extensive investigations, the rate of cholesterol flip-flop and its dependence on the lateral heterogeneity of membranes remain uncertain. In this work, we used atomistic molecular dynamics simulations to sample spontaneous cholesterol flip-flop events in a DPPC:DOPC:cholesterol mixture with heterogeneous lateral distribution of lipids. In addition to an overall flip-flop rate at the time scale of sub-milliseconds, we identified a significant impact of local environment on flip-flop rate. We discuss the atomistic details of the flip-flop events observed in our simulations.

Low- q Bicelles Are Mixed Micelles.

Bicelles are used in many membrane protein studies because they are thought to be more bilayer-like than micelles. We investigated the properties of "isotropic" bicelles by small-angle neutron scattering, small-angle X-ray scattering, fluorescence anisotropy, and molecular dynamics. All data suggest that bicelles with a q value below 1 deviate from the classic bicelle that contains lipids in the core and detergent in the rim. Thus not all isotropic bicelles are bilayer-like.

The molecular mechanism of monolayer-bilayer transformations of lung surfactant from molecular dynamics simulations.

The aqueous lining of the lung surface exposed to the air is covered by lung surfactant, a film consisting of lipid and protein components. The main function of lung surfactant is to reduce the surface tension of the air-water interface to the low values necessary for breathing. This function requires the exchange of material between the lipid monolayer at the interface and lipid reservoirs under dynamic compression and expansion of the interface during the breathing cycle. We simulated the reversible exchange of material between the monolayer and lipid reservoirs under compression and expansion of the interface. We used a mixture of dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol, cholesterol, and surfactant-associated protein C as a functional analog of mammalian lung surfactant. In our simulations, the monolayer collapses into the water subphase on compression and forms bilayer folds. On monolayer reexpansion, the material is transferred from the folds back to the interface. The simulations indicate that the connectivity of the bilayer aggregates to the monolayer is necessary for the reversibility of the monolayer-bilayer transformation. The simulations also show that bilayer aggregates are unstable in the air subphase and stable in the water subphase.

Analytical derivation of thermodynamic characteristics of lipid bilayer from a flexible string model.

We introduce a flexible string model of the hydrocarbon chain and derive an analytical expression for the lateral pressure profile across the hydrophobic core of the membrane. The pressure profile influences the functioning of the embedded proteins and is difficult to measure experimentally. In our model the hydrocarbon chain is represented as a flexible string of finite thickness with a given bending rigidity. In the mean-field approximation we substitute the entropic repulsion between neighboring chains in a lipid membrane by an effective potential. The effective potential is determined self-consistently. The arbitrary chain conformation is expanded over eigenfunctions of the self-adjoint operator of the chain energy density. The lateral pressure distribution across the bilayer is calculated using the path integral technique. We found that the pressure profile is mainly formed by the sum of the partial contributions of a few discrete lowest-energy "eigenconformations." The dependences on temperature and area per lipid of the lateral pressure produced by the hydrocarbon chains are found. We also calculated the chain contribution to the area compressibility modulus and the temperature coefficient of area expansion.

Computer simulations of phase separation in lipid bilayers and monolayers.

Studying phase coexistence in lipid bilayers and monolayers is important for understanding lipid-lipid interactions underlying lateral organization in biological membranes. Computer simulations follow experimental approaches and use model lipid mixtures of simplified composition. Atomistic simulations give detailed information on the specificity of intermolecular interactions, while coarse-grained simulations achieve large time and length scales and provide a bridge towards state-of-the-art experimental techniques. Computer simulations allow characterizing the structure and composition of domains during phase transformations at Angstrom and picosecond resolution, and bring new insights into phase behavior of lipid membranes.

Density based visualization for molecular simulation.

Molecular visualization of structural information obtained from computer simulations is an important part of research work flow. A good visualization technique should be capable of eliminating redundant information and highlight important effects clarifying the key phenomena in the system. Current methods of presenting structural data are mostly limited to variants of the traditional ball-and-stick representation. This approach becomes less attractive when very large biological systems are simulated at microsecond timescales, and is less effective when coarse-grained models are used. Real time rendering of such large systems becomes a difficult task; the amount of information in one single frame of a simulation trajectory is enormous given the large number of particles; at the same time, each structure contains information about one configurational point of the system and no information about statistical weight of this specific configuration. In this paper we report a novel visualization technique based on spatial particle densities. The atomic densities are sampled on a high resolution 3-dimensional grid along a relatively short molecular dynamics trajectory using hundreds of configurations. The density information is then analyzed and visualized using the open-source ParaView software. The performance and capability of the method are demonstrated on two large systems simulated with the MARTINI coarse-grained force field: a lipid nanoparticle for delivering siRNA molecules and monolayers with a complex composition under conditions that induce monolayer collapse.

Simulations of lipid monolayers.

A lipid monolayer lining a boundary between two immiscible phases forms a complex interface with inhomogeneous distribution of forces. Unlike lipid bilayers, monolayers are formed in asymmetric environment and their properties depend strongly on lipid surface density. The monolayer properties are also affected significantly by the representation of the pure interface. Here we give a brief theoretical introduction and describe methods to simulate lipid monolayers starting from force-fields and system setup to reproducing state points on the surface tension (pressure)-area isotherms and transformations between them.

Interaction of pristine and functionalized carbon nanotubes with lipid membranes.

Carbon nanotubes are widely used in a growing number of applications. Their interactions with biological materials, cell membranes in particular, is of interest in applications including drug delivery and for understanding the toxicity of carbon nanotubes. We use extensive molecular dynamics simulations with the MARTINI model to study the interactions of model nanotubes of different thickness, length, and patterns of chemical modification with model membranes. In addition, we characterize the interactions of small bundles of carbon nanotubes with membrane models. Short pristine carbon nanotubes readily insert into membranes and adopt an orientation parallel to the plane of the membrane in the center of the membrane. Larger aggregates and functionalized nanotubes exhibit a range of possible interactions. The distribution and orientation of carbon nanotubes can be controlled by functionalizing the nanotubes. Free energy calculations provide thermodynamic insight into the preferred orientations of different nanotubes and quantify structural defects in the lipid matrix.

Computer simulations of the phase separation in model membranes.

We used computer simulations to investigate the properties of model lipid membranes with coexisting phases. This is relevant for understanding lipid-lipid interactions underlying lateral organization in biological membranes. Molecular dynamics simulations with the MARTINI coarse-grained force field were employed to study lipid bilayers -40 nm in lateral dimension on a 20 micros time scale. The simulations retain near atomic-level detail and lipid chemical specificity, and allow formation of multiple domains of tens of nanometers in size. Using ternary lipid mixtures of saturated and unsaturated lipids and cholesterol, we reproduced the coexistence of the Lalpha/gel phases and the Lo/Ld phases. Phase transformation proceeded by either nucleation or spinodal decomposition. The properties of coexisting phases were characterized in detail, including partial lipid areas, composition, phase boundary and domain registry, based on Voronoi tessellation. We investigated variations of these properties with temperature and surface tension, and compared them to our recent simulations of lipid monolayers of the same size and composition. We found substantial overlap in bilayer and monolayer properties. Increasing the temperature in bilayers produced similar effects as increasing the surface tension in monolayers. This information can be used for interpreting experimental data on model membranes.

Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core.

Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.

Lateral pressure profiles in lipid monolayers.

We have used molecular dynamics simulations with coarse-grained and atomistic models to study the lateral pressure profiles in lipid monolayers. We first consider simple oillair and oil/water interfaces, and then proceed to lipid monolayers at air/water and oil/water interfaces. The results are qualitatively similar in both atomistic and coarse-grained models. The lateral pressure profile in a monolayer is characterized by a headgroup/water pressure-interfacial tension-chain pressure pattern. In contrast to lipid bilayers, the pressure decreases towards the chain free ends. An additional chain/air tension peak is present in monolayers at the air/water interface. Lateral pressure profiles are calculated for monolayers of different lipid composition under varying surface tension. Increasing the surface tension suppresses both pressure peaks and widens the interfacial tension in monolayers at the oil/water interface, and mainly suppresses the chain pressure in monolayers at the air/water interface. In monolayers in the liquid-condensed phase, the pressure peaks split due to ordering. Variation of lipid composition leads to noticeable changes in all regions of the pressure profile at a fixed surface tension.