Simulations Of Lipid Bilayers Research Articles

Cholesterol-Induced Membrane Organization Promotes Influenza Virus Binding

The influenza virus attaches to a cell surface by binding sialic-acid containing receptors with the viral ligand hemagglutinin. Stable viral binding requires more than one viral ligand- host receptor interaction to simultaneously occur. Thus, binding can be modulated by the spatial distribution of target receptors in the host membrane. We show that membrane sterols critically control viral attachment and propose that sterols alter the nanoscale clustering of viral receptors to facilitate binding. Using single virus fluorescence microscopy, we demonstrated that viral binding is dependent on the cholesterol content of the target membrane. Fluorescently labeled viral particles preferentially bound synthetic membranes supplemented with increasing amounts of cholesterol (0-40 mol%). Other sterols exhibited a similar effect on binding, independent of their ability to support liquid-liquid phase separation. To develop a molecular explanation for cholesterol's effect, we ran a series of course grained molecular dynamics simulations of lipid bilayers containing the viral receptor, disialoganglioside GD1a. While simulated GD1a molecules self-associated independent of cholesterol, the dissociation rate between pairs of GD1a molecules was a function of bilayer cholesterol concentration. Additionally, cholesterol increased the order parameter of simulated GD1a lipid tails. We suggest that, by preordering the viral receptor in its monomeric state, cholesterol lowers the entropic penalty of receptor association. This in turn promotes the formation of GD1a multimers and increases the influenza virus binding avidity of the lipid bilayer. Our findings assign a critical role to the host cellular membrane in viral infectivity and reveal sterol-dependent membrane organization not associated with phase separation.

Water permeation across artificial I-quartet membrane channels: from structure to disorder.

Artificial water channels (AWCs) have been designed for water transport across membranes with the aim to mimic the high water permeability observed for biological systems such as aquaporins (∼108-109 water molecules per s per channel), as well as their selectivity to reject ion permeation at the same time. Recent works on designed self-assembling alkylureido-ethylimidazole compounds forming imidazole-quartet channels (I-quartets), have shown both high water permeability and total ionic-rejection. I-quartets are thus promising candidates for further development of AWCs. However, the molecular mechanism of water permeation as well as I-quartet organization and stability in a membrane environment need to be fully understood to guide their optimal design. Here, we use a wide range of all-atom molecular dynamics (MD) simulations and their analysis to understand the structure/activity relationships of the I-quartet channels. Four different types with varying alkyl chain length or chirality have been studied in a complex fully hydrated lipid bilayer environment at both microsecond and nanosecond scale. Microsecond simulations show two distinct behaviors; (i) two out of four systems maintain chiral dipolar oriented water wires, but also undergo a strong reorganization of the crystal shape, (ii) the two other I-quartet channels completely lose the initial organization, nonetheless keeping a water transport activity. Short MD simulations with higher time resolution were conducted to characterize the dynamic properties of water molecules in these model channels and provided a detailed hypothesis on the molecular mechanism of water permeation. The ordered confined water was characterized with quantitative measures of hydrogen-bond life-time and single particle dynamics, showing variability among I-quartet channels. We will further discuss the underlying assumptions, currently based on self-aggregation simulations and crystal patches embedded in lipid bilayer simulations and attempt to describe possible alternative approaches to computationally capture the water permeation mechanism and the self-assembly process of these AWCs.

Electropore Formation in Mechanically Constrained Phospholipid Bilayers.

Molecular dynamics simulations of lipid bilayers in aqueous systems reveal how an applied electric field stabilizes the reorganization of the water-membrane interface into water-filled, membrane-spanning, conductive pores with a symmetric, toroidal geometry. The pore formation process and the resulting symmetric structures are consistent with other mathematical approaches such as continuum models formulated to describe the electroporation process. Some experimental data suggest, however, that the shape of lipid electropores in living cell membranes may be asymmetric. We describe here the axially asymmetric pores that form when mechanical constraints are applied to selected phospholipid atoms. Electropore formation proceeds even with severe constraints in place, but pore shape and pore formation time are affected. Since lateral and transverse movement of phospholipids may be restricted in cell membranes by covalent attachments to or non-covalent associations with other components of the membrane or to membrane-proximate intracellular or extracellular biomolecular assemblies, these lipid-constrained molecular models point the way to more realistic representations of cell membranes in electric fields.

Permeabilizing Phospholipid Bilayers with Non-normal Electric Fields

Since 2003, molecular dynamics simulations of lipid bilayers have provided valuable insights into the mechanisms underlying electropermeabilization (electroporation)-an electric field-induced increase in the permeability of biological membranes. The convention in these studies has been to apply the electric field normal to the plane of the membrane. In a typical electroporation application, however, where the electric field is reasonably uniform and unidirectional, the field is perpendicular to the membrane only at a few locations-for spherical cells only at the poles of the cells along the axis defined by the direction of the electric field. Everywhere else on the cell surface the field is applied at an angle that is oblique to the plane of the membrane. On a microscopic level, the invaginations and protrusions that characterize a living cell membrane also present many angles to the applied electric field. Here we report the results of molecular dynamics simulations of lipid electropore formation when the electric field is not normal to the membrane surface, which show that the tangential component of the field has a small but non-zero effect.

Simulation of Linoleoyl-Containing Pure Lipid Bilayer and Soybean Plasma Membranes

Molecular dynamics (MD) simulations have been extensively used to study lipid membranes in addition to experimental studies as they help better understand membranes in the atomic level. Computational models of bacterial (E. coli) membranes have been developed and applied to study the antimicrobial peptide proteins. Plant membranes are less frequently studied compared to the bacterial membrane. In this work, we will present the soybean plasma membranes models. The compositions of cell plasma membranes of soybean vary depending on the species, stage of development, and the part of the plant. The two parts of the plant that we study are the hypocotyl and the root. Each model consists of 100 lipids per leaflet, with the composition based on the weighted and averaged values from past experimental studies. Specifically, the hypocotyl membrane contains 7 types of unsaturated phospholipids and two types of sterols, while the root membrane contains 8 types of phospholipids and two types of sterols. All types of phospholipids in soybean contains the 18:2 (cis Δ9, 12) linoleoyl tail which was not well studied before, therefore, the simulations on the pure 18:0/18:2 and 18:2/18:2 phosphocholine (PC) lipid bilayers are also performed. The structural properties such as surface area per lipid, bilayer thicknesses, order parameters, and spin-lattice relaxation time are analyzed for all membranes. Moreover, the analyses of the sterols tilt angle distributions, hydrogen bonding, and clustering are also conducted for the soybean membranes. The structural properties of pure bilayers agree well with NMR experimental data validate the accuracy of 18:2 linoleoyl-containing lipids, based on which the soybean membrane models also result in reasonable structural properties. These results imply that the two soybean membrane models are realistic, and can facilitate the further study of soybean and other plant membranes.

Influenza Binding Avidity Governed by Sterol-Dependent Ganglioside Dynamics

Influenza virions attach to host membranes by binding cell-surface glycans containing sialic acid, initiating the viral life cycle and infecting cells. While the primary determinant of viral binding is thought to be the glycan chemical structure, single interactions between a viral hemagglutinin binding site and a sialic acid are weak, so binding specificity is presumed to occur via multivalent interactions. Here, we show that target membrane composition can alter influenza binding while total receptor content is held constant. We hypothesize that this effect occurs by changing the spatial distribution of target receptors. Viral binding was quantitated by monitoring individual viruses binding to synthetic supported lipid bilayers using fluorescence microscopy. Adding cholesterol to target bilayers increased binding in a dose-dependent manner. Furthermore, membranes containing 5-cholesten-3-one, an oxidized variant of cholesterol, displayed significantly higher binding avidity compared to membranes supplemented with an equal amount of cholesterol. When sterol mole fractions were held constant, viral target receptor concentration and binding exhibited a sigmoidal relationship, confirming the multivalent nature of virus receptor binding. The cooperativity required for binding established the potential for local receptor concentration to regulate membrane binding affinity. To develop a physical model for sterol-induced changes in the lateral organization of GD1a receptors, we performed coarse grained molecular dynamics simulations of target lipid bilayers. Irrespective of sterol content, GD1a receptors showed strong tendencies to self-associate. The inclusion of cholesterol in membranes increased pairwise receptor contacts, suggesting that sterol addition stabilized target receptor clustering. This observation supports the hypothesis that sterols can modulate membrane nanoscale organization and thus alter viral binding avidity, illuminating a previously underappreciated mechanism by which target membrane context can influence influenza infectivity.

Stress Propagation through Biological Lipid-Bilayers Revealed by Atomistic and Coarse-Grained Simulations

Membrane tension has been shown to play various critical roles in cell signaling. We here asked if and how pulses of local stress dynamically propagate through membranes, and propose this mechanism as a novel way of quickly propagating signals along the membrane.In both atomistic and coarse-grained MARTINI molecular dynamics simulations of biological lipid-bilayers, we observed short stress pulses to very efficiently propagate laterally at a velocity of the order of km/s, in close agreement with the expected speed of sound. The temperature dependence of pulse propagation shows tendencies very comparable to analogous experiments [1,2,3], with insightful differences between the atomistic and coarse-grained simulations. Remarkably, the propagation of the lateral stress was damped at length scales in the ∼100 nm range. Our data supports the notion of lateral stress propagation through membranes as a potential ultrafast way of short-range signal propagation in biology [4].[1] W. Schrader, et al. J. Phys. Chem. B. 106:6581-6586 (2002)[2] S. Shrivastava S and MF Schneider. J. R. Soc. Interface 11:20140098 (2014).[3] J. Kappler and R. Netz. EPL. 112: 19002 (2015)[4] C. Aponte-Santamaria and F. Grater. In preparation

Molecular dynamics simulations of ternary lipid bilayers containing plant sterol and glucosylceramide

An atomic-level molecular dynamics simulation was carried out to study the effects of a plant sterol (sitosterol) and glucosylceramide (GlcCer) on a 1,2-dilinoleoylposphocholine (DLiPC) membrane. Initially, a membrane containing 50mol% sitosterol was compared with that containing the same ratio of cholesterol. These simulations showed differential condensing and ordering effects of sitosterol and cholesterol, with cholesterol being slightly more efficient than sitosterol in packing the membrane more tightly to a liquid ordered phase. By incorporation of 9.3% GlcCer on DLiPC/sterol membrane no notable change was observed in terms of area per lipid, bilayer thickness, order parameter and lateral diffusion. Some clusters of GlcCer/sterol were observed at higher ratio of GlcCer (15.5%), supporting the existence of GlcCer/sitosterol-enriched Lo-domains on the nanometer scale in the plant lipid mixture.

Principal Component Analysis of Lipid Molecule Conformational Changes in Molecular Dynamics Simulations.

Molecular dynamics simulations of lipid bilayers are ubiquitous nowadays. Usually, either global properties of the bilayer or some particular characteristics of each lipid molecule are evaluated in such simulations, but the structural properties of the molecules as a whole are rarely studied. Here, we show how a comprehensive quantitative description of conformational space and dynamics of a single lipid molecule can be achieved via the principal component analysis (PCA). We illustrate the approach by analyzing and comparing simulations of DOPC bilayers obtained using eight different force fields: all-atom generalized AMBER, CHARMM27, CHARMM36, Lipid14, and Slipids and united-atom Berger, GROMOS43A1-S3, and GROMOS54A7. Similarly to proteins, most of the structural variance of a lipid molecule can be described by only a few principal components. These major components are similar in different simulations, although there are notable distinctions between the older and newer force fields and between the all-atom and united-atom force fields. The DOPC molecules in the simulations generally equilibrate on the time scales of tens to hundreds of nanoseconds. The equilibration is the slowest in the GAFF simulation and the fastest in the Slipids simulation. Somewhat unexpectedly, the equilibration in the united-atom force fields is generally slower than in the all-atom force fields. Overall, there is a clear separation between the more variable previous generation force fields and significantly more similar new generation force fields (CHARMM36, Lipid14, Slipids). We expect that the presented approaches will be useful for quantitative analysis of conformations and dynamics of individual lipid molecules in other simulations of lipid bilayers.

Lipid-Lipid Coupling to Membrane Curvature by Simulation and NMR

The material properties of a bilayer must be determined by the chemical structure of its lipid components and the interactions between them. The preferred (“spontaneous”) curvature of a lipid leaflet is one of the most interesting properties as it is the most direct connection between the stability of leaflet structures and lipid chemistry. For example, the curved lipid hexagonal phase is stabilized by phosphatidylethanolamine head groups.Nuclear magnetic resonance spectroscopy and molecular dynamics can probe structure on the molecular level, but relating these techniques to material properties requires new models and theoretical methods.In this work, we use computer simulation of lipid bilayers (in particular, sphingomyelin and DPPC) to predict the impact of chemical structure on spontaneous curvature. Then, NMR applied to small vesicles measures how lipid identity controls population of the inner and outer leaflet, reflecting the lipid's spontaneous curvature. Simulations predict that at low concentration of sphingomyelin, strong coupling between lipids determines this behavior in a non-linear way, in contrast to DPPC.

A Parameterization of Cholesterol for Mixed Lipid Bilayer Simulation within the Amber Lipid14 Force Field.

The Amber Lipid14 force field is expanded to include cholesterol parameters for all-atom cholesterol and lipid bilayer molecular dynamics simulations. The General Amber and Lipid14 force fields are used as a basis for assigning atom types and basic parameters. A new RESP charge derivation for cholesterol is presented, and tail parameters are adapted from Lipid14 alkane tails. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers are simulated at a range of cholesterol contents. Experimental bilayer structural properties are compared with bilayer simulations and are found to be in good agreement. With this parameterization, another component of complex membranes is available for molecular dynamics with the Amber Lipid14 force field.

Cis and trans unsaturated phosphatidylcholine bilayers: A molecular dynamics simulation study

Trans unsaturated lipids are uncommon in nature. In the human diet, they occur as natural products of ruminal bacteria or from industrial food processing like hydrogenation of vegetable oils. Consumption of trans unsaturated lipids has been shown to have a negative influence on human health; in particular, the risk of cardiovascular disease is higher when the amount of trans unsaturated lipids in the diet is elevated. In this study, we first performed quantum mechanical calculations to specifically and accurately parameterize cis and trans mono-unsaturated lipids and subsequently validated the newly derived parameter set. Then, we carried out molecular dynamics (MD) simulations of lipid bilayers composed of cis or trans unsaturated lipids with and without cholesterol. Our results show that trans mono-unsaturated chains are more flexible than cis mono-unsaturated chains due to lower barriers for rotation around the single bonds next to the trans double bond than those next to the cis double bond. In effect, interactions between cholesterol and trans unsaturated chains are stronger than cis unsaturated chains, which results in a higher ordering effect of cholesterol in trans unsaturated bilayers.

How Tolerant are Membrane Simulations with Mismatch in Area per Lipid between Leaflets?

Difficulties in estimating the correct number of lipids in each leaflet of complex bilayer membrane simulation systems make it inevitable to introduce a mismatch in lipid packing (i.e., area per lipid) and thus alter the lateral pressure of each leaflet. To investigate potential impacts of such mismatch on simulation results, we performed molecular dynamics simulations of saturated and monounsaturated lipid bilayers with and without gramicidin A or WALP23 at various mismatches by adjusting the number of lipids in the lower leaflet from no mismatch to a 25% reduction compared to that in the upper leaflet. All simulations were stable under the constant pressure barostat, but the mismatch induces asymmetric lipid packing between the leaflets, so that the upper leaflet becomes more ordered, and the lower leaflet becomes less ordered. The mismatch impacts on various bilayer properties are mild up to 5–10% mismatch, and bilayers with fully saturated chains appear to be more prone to these impacts than those with unsaturated tails. The nonvanishing leaflet surface tensions and the free energy derivatives with respect to the bilayer curvature indicate that the bilayer would be energetically unstable in the presence of mismatch. We propose a quantitative criterion for allowable mismatch based on the energetics derived from a continuum elastic model, which grows as a square root of the number of the lipids in the system. On the basis of this criterion, we infer that the area per lipid mismatch up to 5% would be tolerable in various membrane simulations of reasonable all-atom system sizes (40–160 lipids per leaflet).

Constant-pH MD Simulations of an Oleic Acid Bilayer.

Oleic acid is a simple molecule with an aliphatic chain and a carboxylic group whose ionization and, consequently, intermolecular interactions are strongly dependent on the solution pH. The titration curve of these molecules was already obtained using different experimental methods, which have shown the lipid bilayer assemblies to be stable between pH 7.0 and 9.0. In this work, we take advantage of our recent implementations of periodic boundary conditions in Poisson-Boltzmann calculations and ionic strength treatment in simulations of charged lipid bilayers, and we studied the ionization dependent behavior of an oleic acid bilayer using a new extension of the stochastic titration constant-pH MD method. With this new approach, we obtained titration curves that are in good agreement with the experimental data. Also, we were able to estimate the slope of the titration curve from charge fluctuations, which is an important test of thermodynamic consistency for the sampling in a constant-pH MD method. The simulations were performed for ionizations up to 50%, because an experimentally observed macroscopic transition to micelles occurs above this value. As previously seen for a binary mixture of a zwitterionic and an anionic lipid, we were able to reproduce experimental results with simulation boxes usually far from neutrality. This observation further supports the idea that a charged membrane strongly influences the ion distribution in its vicinity and that neutrality is achieved significantly far from the bilayer surface. The good results obtained with this extension of the stochastic titration constant-pH MD method strongly supports its usefulness to sample the coupling between configuration and protonation in these types of biophysical systems. This method stands now as a powerful tool to study more realistic lipid bilayers where pH can influence both the lipids and the solutes interacting with them.

Experimental Assessment of Tilt-Dependent Thermal Fluctuations in Lipid Bilayers

For length scales shorter than several membrane thicknesses, many molecular dynamics simulations of single component lipid bilayers have reported significant deviations in the measured height-height fluctuation spectrum compared to predictions from the Helfrich free energy. Recently, others have posited membrane models that depend on molecular tilt and that have been shown to be consistent with the aforementioned deviations. We present the first experimental support for a tilt-dependent theory for biomembrane mechanics. X-ray scattering from a liquid crystalline stack of oriented fluid phase lipid bilayers was collected and compared to the predictions of tilt-dependent and tilt-independent membrane models. Both tilt-dependent and -independent models satisfactorily fit the X-ray data dominated by in-plane correlation lengths much greater than membrane thickness (> 100 A), but only a tilt-dependent model accounts for X-ray data primarily attributable to shorter length correlations. By fitting the measured X-ray scattering intensity, both the bending modulus Kc = 8.3 ± 0.6×10−20 J and the tilt modulus Kθ = 95 ± 7 mN/m were determined for DOPC lipid bilayers at 30°C. Our experimental results support the enrichment of the classic Helfrich continuum model to include molecular tilt. Since the size of many biomembrane related molecules is similar to tilt-dependent length scales, the tilt modulus may be highly sensitive to various membrane-biomolecule interactions.

An Investigation of the Interaction between Melittin and a Model Lipid Bilayer

The recent emergence of multi-drug resistant bacteria has rendered manycommon antibiotics ineffective and therefor novel drugs are needed. Antimi-crobial peptides have proven applicable in this aspect as they can formself-assembled pore structures in the lipid bilayer of bacterial cells. Theexact pore formation is shrouded in controversy and largely unknown as it isdifficult to determine empirically. The development of coarse grain force fieldsin molecular dynamic simulations enables longer timescales and extendedsimulation systems. This allows for the simulation of lipid bilayer self-assembly and bilayer-antimicrobial peptide interactions. The present workfocuses on the antimicrobial peptide, melittin, induced pore formation and isaimed towards determining the mechanism and pore structure

Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

Molecular dynamics simulations of membrane proteins.

Membrane protein structures are underrepresented in the Protein Data Bank (PDB) due to difficulties associated with expression and crystallization. As such, it is one area where computational studies, particularly Molecular Dynamics (MD) simulations, can provide useful additional information. Recently, there has been substantial progress in the simulation of lipid bilayers and membrane proteins embedded within them. Initial efforts at simulating membrane proteins embedded within a lipid bilayer were relatively slow and interactive processes, but recent advances now mean that the setup and running of membrane protein simulations is somewhat more straightforward, though not without its problems. In this chapter, we outline practical methods for setting up and running MD simulations of a membrane protein embedded within a lipid bilayer and discuss methodologies that are likely to contribute future improvements.

Testing procedures for extracting fluctuation spectra from lipid bilayer simulations.

To address concerns about how to obtain the height-height spectrum from simulations of biomembranes, we emulated the fluctuations in real space using exact input spectra. Two different methods that have given different results in the literature were then used to extract spectra from the emulated fluctuations that were then compared to the exact input spectra. A real space method shows systematic, but small deviations attributed to splines introducing an artifactual filter. A direct Fourier method obtains accurate results when the in-plane placement of the emulated particles is uncorrelated with the out-of-plane undulations, but systematic underestimates occur when the particle placement is more realistically correlated with the undulations. Although quantitative corrections cannot be estimated from our one-dimensional model, the results are qualitatively consistent with the direct Fourier method underestimating the 1/q(2) spectral dependence that is characteristic of a tilt degree of freedom in simulations.

Molecular Dynamics Simulation of Inverse-Phosphocholine Lipids

We have performed molecular dynamics simulations of lipid bilayers composed of inverse-phosphatidylcholines (CPe), an analog of phosphatidylcholine (PC) with the choline group directly bound to the glycerol backbone and the phosphate group freely protruding into the water phase. Synthetic phospholipids with the CPe headgroup have been proposed for use as drug delivery liposomes. Our simulation results show that the CPe lipids were characterized by a larger area per lipid molecule than the PC lipids. This can partly explain experimental results that show a higher permeability of small solutes through the membranes of liposomes composed of them. Unlike the PC headgroup, the CPe headgroup was found not to bind sodium ions at the water membrane interface. Both lipid types were found to bind calcium ions but do not bind potassium ions. Inversion of the choline group was found to decrease hydration of the membrane in the carbonyl region of the bilayer as well as hydration of the choline group. From analyzing the water ordering in our simulation, we determined that the orientation of the water layer next to the CPe membrane is effectively inverted with respect to the water ordering of the PC membrane, possibly affecting interaction with biomembranes encountered in drug delivery. Due to changes in ion binding, charge group distribution, and water orientation, the electrostatic potential profiles across the lipid bilayer of CPe membranes were found to differ considerably from those of PC membranes. This is a possible explanation of the experimentally observed changes in the charged solute permeability.