A systematic molecular dynamics simulation study of temperature dependent bilayer structural properties

Although lipid force fields (FFs) used in molecular dynamics (MD) simulations have proved to be accurate, there has not been a systematic study on their accuracy over a range of temperatures. Motivated by the X-ray and neutron scattering measurements of common phosphatidylcholine (PC) bilayers (Ku?erka et al. BBA. 1808: 2761, 2011), the CHARMM36 (C36) FF accuracy is tested in this work with MD simulations of six common PC lipid bilayers over a wide range of temperatures. The calculated scattering form factors and deuterium order parameters from the C36 MD simulations agree well with the X-ray, neutron, and NMR experimental data. There is excellent agreement between MD simulations and experimental estimates for the surface area per lipid, bilayer thickness (D_B), hydrophobic thickness (D_C), and lipid volume (V_L). The only minor discrepancy between simulation and experiment is a measure of (D_B − D_HH) / 2 where D_HH is the distance between the maxima in the electron density profile along the bilayer normal. Additional MD simulations with pure water and heptane over a range of temperatures provide explanations of possible reasons causing the minor deviation. Overall, the C36 FF is accurate for use with liquid crystalline PC bilayers of varying chain types and over biologically relevant temperatures.     

Outer membrane proteins: comparing X-ray and NMR structures by MD simulations in lipid bilayers

The structures of three bacterial outer membrane proteins (OmpA, OmpX and PagP) have been determined by both X-ray diffraction and NMR. We have used multiple (7 × 15 ns) MD simulations to compare the conformational dynamics resulting from the X-ray versus the NMR structures, each protein being simulated in a lipid (DMPC) bilayer. Conformational drift was assessed via calculation of the root mean square deviation as a function of time. On this basis the ‘quality’ of the starting structure seems mainly to influence the simulation stability of the transmembrane β-barrel domain. Root mean square fluctuations were used to compare simulation mobility as a function of residue number. The resultant residue mobility profiles were qualitatively similar for the corresponding X-ray and NMR structure-based simulations. However, all three proteins were generally more mobile in the NMR-based than in the X-ray simulations. Principal components analysis was used to identify the dominant motions within each simulation. The first two eigenvectors (which account for >50% of the protein motion) reveal that such motions are concentrated in the extracellular loops and, in the case of PagP, in the N-terminal α-helix. Residue profiles of the magnitude of motions corresponding to the first two eigenvectors are similar for the corresponding X-ray and NMR simulations, but the directions of these motions correlate poorly reflecting incomplete sampling on a ∼10 ns timescale.     

Conjugated double bonds in lipid bilayers: a molecular dynamics simulation study

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is attributed to have the anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be responsible for the anti-obesity effects. Since dietary CLA are incorporated into membrane phospholipids, we have used Molecular Dynamics (MD) simulations to investigate the comparative effects of the two isomers on lipid bilayer structure. Specifically, simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed. Force field parameters for the torsional potential of double bonds were obtained from ab initio calculations. From the MD trajectories we calculated and compared structural properties of the two lipid bilayers, including areas per molecule, density profiles, thickness of bilayers, tilt angle of tail chains, order parameters profiles, radial distribution function (RDF) and lateral pressure profiles. The main differences found between bilayers of the two CLA isomers, are (1) the order parameter profile for C9T11 has a dip in the middle of sn-2 chain while the profile for T10C12 has a deeper dip close to terminal of sn-2 chain, and (2) the lateral pressure profiles show differences between the two isomers. Our simulation results reveal localized physical structural differences between bilayers of the two CLA isomers that may contribute to different biological effects through differential interactions with membrane proteins or cholesterol.     

Effect of magnesium sulfate in oxidized lipid bilayers properties by using molecular dynamics

Magnesium sulfate (MgSO₄) has been used as a protector agent for many diseases related to oxidative stress. The effect of MgSO₄ on the oxidized lipid bilayer has not yet been studied using molecular dynamics calculations. In this work, the effects of oxidation were evaluated by using a POPC membrane model at different concentrations of its aldehyde (-CHO) and hydroperoxide (-OOH) derivatives with and without MgSO₄. Several quantitative and qualitative properties were evaluated, such as membrane thickness, area per lipid, area compressibility modulus, snapshots after simulation finish, density distributions, time evolutions of oxidized group positions, and radial distributions of oxidized group concerning Mg. Results indicate that in the absence of MgSO₄ the mobility of oxidized groups, particularly –CHO, toward the surface interface is high. At a low oxidation level of the bilayer there is an increase in the compressibility modulus as compared to the unoxidized bilayer. MgSO₄, at a low oxidation level, tends to lessen the oxidation effects by lowering the dispersion in the distribution of oxidized species toward the membrane surface and the water region. However, MgSO₄ does not change the trends of decreasing membrane thickness and area compressibility modulus and increasing area per lipid upon oxidation. In this regard, MgSO₄ diminishes the electrostatic long-distance attractive interactions between the oxidized groups and the charged headgroups of the interface, owing to the Mg⁺² and SO₄^-2 screening effects and an electrostatic stabilization of the headgroups, preventing the pore formation, which is well-known to occur in oxidized membranes.     

Interaction between K+ channel gate modifier hanatoxin and lipid bilayer membranes analyzed by molecular dynamics simulation

Hanatoxin (HaTx) is an ellipsoidal-shaped peptide that binds to the voltage sensor of voltage-dependent channels. Of physicochemical interest, HaTx has a “ring” of charged residues around its periphery and a hydrophobic protrusion. It has previously been postulated that HaTx binds to and functions on the surface of membranes, but a recent fluorescent-quenching study has implied a fairly deep positioning of HaTx in the lipid bilayer membrane. We carried out numerous molecular dynamic simulations of HaTx1, a well-studied variant of HaTx, in fully hydrated phospholipid bilayers. The system reproduced the surface-binding mode of HaTx1, in which HaTx1 resided in the extracellular side (outer) of the water/membrane interface with the hydrophobic patch of HaTx1 facing the membrane interior. On the other hand, analyses with various parameter settings suggested that the surface-binding mode was unstable because of the substantial attractive electrostatic force between HaTx1 and the lipid head groups of the inner (opposite) leaflet. Compared with this electrostatic force, the energetic cost for membrane deformation involving meniscus formation appeared to be small. In an attempt to interpret the quenching data, we consider the possibility of dimpling (meniscus formation) that brings HaTx1 inward (only ~0.7–0.8 nm above the bilayer center), while accounting for the flexibility of both leaflets of the membrane and the long-range interaction between positively charged residues of the membrane-bound peptide and the polar head groups of the opposite leaflet of the membrane. It is suggested that molecular dynamics simulations taking into account the flexibility of the membrane surface is potentially useful in interpreting the fluorescence-quenching data.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Structure and dynamics of the antifungal molecules Syringotoxin-B and Syringopeptin-25A from molecular dynamics simulation

The phytopathogen Pseudomonas syringae pv. syringae produces toxic cyclic lipodepsipeptides (CLPs): nona-peptides and syringopeptins. All CLPs inhibit the growth of many fungal species, including human pathogens, although different fungi display different degrees of sensitivity. The best studied CLPs are Syringomycin-E (SR-E), Syringotoxin-B (ST-B) and Syringopeptin-25A (SP-25A). Their biological activity is affected by membrane composition and their structural differences. We previously (Matyus et al. in Eur Biophys J 35:459–467, 2006) reported the molecular features and structural preferences of SR-E in water and octane environments. Here we investigate in atomic detail the molecular features of the two other main CLP components, ST-B and SP-25A, in water and octane by 200 ns molecular dynamics simulations (MD), using distance restraints derived from NMR NOE data (Ballio et al. in Eur J Biochem 234:747–758, 1995). We have obtained three-dimensional models of ST-B and SP-25A CLPs in different environments. These models can now be used as a basis to investigate the interactions of ST-B and SP-25A with lipid membranes an important further step towards a better understanding of the antifungal and antibacterial activity of these peptides. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular aspects of the interaction between amphotericin B and a phospholipid bilayer: molecular dynamics studies

Amphotericin B (AmB) is a polyene macrolide antibiotic used to treat systemic fungal infections. The molecular mechanism of AmB action is still only partly characterized. AmB interacts with cell-membrane components and forms membrane channels that eventually lead to cell death. The interaction between AmB and the membrane surface can be regarded as the first (presumably crucial) step on the way to channel formation. In this study molecular dynamics simulations were performed for an AmB–lipid bilayer model in order to characterize the molecular aspects of AmB–membrane interactions. The system studied contained a box of 200 dimyristoylphosphatidylcholine (DMPC) molecules, a single AmB molecule placed on the surface of the lipid bilayer and 8,065 water molecules. Two molecular dynamics simulations (NVT ensemble), each lasting 1 ns, were performed for the model studied. Two different programs, CHARMM and NAMD2, were used in order to test simulation conditions. The analysis of MD trajectories brought interesting information concerning interactions between polar groups of AmB and both DMPC and water molecules. Our studies show that AmB preferentially took a vertical position, perpendicular to the membrane surface, with no propensity to enter the membrane. Our finding may suggest that a single AmB molecule entering the membrane is very unlikely.Figure The figure presents the whole structure of the system simulated—starting point. AmB is presented as a space-filling model, DMPC molecules—green sticks, water molecules—red sticks     

Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives — A molecular dynamics study

Amphotericin B (AmB) is an effective but very toxic antifungal antibiotic. In our laboratory a series of AmB derivatives of improved selectivity of action was synthesized and tested. To understand molecular basis of this improvement, comparative conformational studies of amphotericin B and its two more selective derivatives were carried out in an aqueous solution and in a lipid membrane. These molecular simulation studies revealed that within a membrane environment the conformational behavior of the derivatives differs significantly from the one observed for the parent molecule. Possible reasons for such a difference are analyzed. Furthermore, we hypothesize that the observed conformational transition within the polar head of AmB derivatives may lead to destabilization of antibiotic-induced transmembrane channels. Consequently, the selective toxicity of the derivatives should increase as ergosterol-rich liquid-ordered domains are more rigid and conformationally ordered than their cholesterol-containing counterparts, and as such may better support less stable channel structure.     

Binding and insertion of alpha-helical anti-microbial peptides in POPC bilayers studied by molecular dynamics simulations

We have performed molecular dynamics simulations of the interactions of two alpha-helical anti-microbial peptides, magainin2 and its synthetic analog of MSI-78, with palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers. We used various initial positions and orientations of the peptide with respect to the lipid bilayer, including a surface-bound state parallel to the interface, a trans-membrane state, and a partially inserted state. Our 20 ns long simulations show that both magainin2 and MSI-78 are most stable in the lipid environment, with the peptide destabilized to different extents in both aqueous and lipid/water interfacial environments. We found that there are strong specific interactions between the lysine residues of the peptides and the lipid head-group regions. MSI-78, owing to its large number of lysines, shows better binding characteristics and overall stability when compared to magainin2. We also find that both peptides destabilize the bilayer environment, as observed by the increase in lipid tail disorder and the induction of local curvature on the lipid head-groups by the peptides. From all the simulations, we conclude that the hydrogen bonding interactions between the lysines of the peptides and the oxygens of the polar lipid head-groups are the strongest and determine the overall peptide binding characteristics to the lipids.     

The transmembrane domain of Neu in a lipid bilayer: molecular dynamics simulations

The results of full-atom molecular dynamics simulations of the transmembrane domains (TMDs) of both native, and Glu⁶⁶⁴-mutant (either protonated or unprotonated) Neu in an explicit fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer are presented. For the native TMD peptide, a 10.05 ns trajectory was collected, while for the mutant TMD peptides 5.05 ns trajectories were collected for each. The peptides in all three simulations display stable predominantly -helical hydrogen bonding throughout the trajectories. The only significant exception occurs near the C-terminal end of the native and unprotonated mutant TMDs just outside the level of the lipid headgroups, where -helical hydrogen bonding develops, introducing a kink in the backbone structure. However, there is no indication of the formation of a bulge within the hydrophobic region of either native or mutant peptides. Over the course of the simulation of the mutant peptide, it is found that a significant number of water molecules penetrate the hydrophobic region of the surrounding lipid molecules, effectively hydrating Glu⁶⁶⁴. If the energy cost of such water penetration is significant enough, this may be a factor in the enhanced dimerization affinity of Glu⁶⁶⁴-mutant Neu.     

Interaction of lysophosphatidylcholine with phosphatidylcholine bilayers. A photo-physical and NMR study

Several photo-physical methods together with ³¹P-NMR have been used to investigate the effect of lysophosphatidylcholine on phosphatidylcholine bilayers. ³¹P-NMR shows that the permeability of the vesicle to Eu³⁺ increases sharply above approx. 40% lysophosphatidylcholine: fluorescence-quenching studies also show this type of behavior. Similar sharp changes in vesicle properties are observed via the photo-physical technique at this lysophosphatidylcholine/phosphatidylcholine composition. Fluorescence spectra of pyrene and pyrene carboxaldehyde show that increasing lysophosphatidylcholine composition increases the polarity of the environments of these probes up to 40% lysocompound. Above this composition the photo-physical properties of the probes slowly revert to those characteristic of the micellar lyso-compound. The pyrene fluorescence lifetime, the fine structure of the fluorescence, and the case of formation of pyrene excimer in these bilayer mixtures suggest that pyrene complexes weakly with the charged nitrogen of the choline group of the phosphatidylcholine and that the physical state of the system has a striking effect on this complexation process. Similar experiments with simple quaternary compounds lend strong support to this suggestion. The studies monitor in several ways the effect of bilayer composition on movement of molecules in these systems. The degree or site of solubilization of carcinogens is also uniquely affected by composition.     

Purification of squid rhodopsin and reassembly into lipid bilayers

Rhodopsin from squid photoreceptor membranes was solubilized in octyl glucoside and purified to a single band on SDS-polyacrylamide gels of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 46 000. Purified rhodopsin was recombined with phospholipids to form vesicles by detergent dialysis. Spectroscopic analysis of the rhodopsin-lipid vesicles showed that the interconversion between acid and basic metarhodopsin had a $\text{p}\text{K}$ of 8. Furthermore, rhodopsin in the vesicles could be photoregenerated from metarhodopsin in solutions of either neutral or alkaline pH. These two spectroscopic properties are comparable to those for rhodopsin in photoreceptor membranes. The results indicate that the native conformation of rhodopsin is preserved during purification and after recombination with phospholipids into vesicles. This preparation is, therefore, an active starting point for functional reconstitution studies.     

Setting up and running molecular dynamics simulations of membrane proteins

Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the protein(s) on a widely spaced grid and then 'shrinking' the grid until the bilayer with the protein has the desired density, with lipids neatly packed around the protein. When starting from a grid based on a single lipid structure, or several potentially different lipid structures (method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD simulations of membrane proteins.     

Lipid bilayer polypeptide interactions studied by molecular dynamics simulation

A model membrane with a polypeptide alpha-helix inserted has been simulated by molecular dynamics at a temperature well above the gel/liquid crystalline phase transition temperature. Order parameters of the lipids and other equilibrium and dynamic quantities have been calculated. Three systems, polyglycine constrained into an alphahelical configuration, glycophorin with similarly conformationally constrained backbone and finally glycophorin free to change its backbone conformation, have been studied. In all cases there was an ordering of the chains close to the helix. This effect was, however, much smaller for glycophorin with its rather bulky side chains than for polyglycine. The dynamics of the lipids were affected by the neighbouring helix, not drastically however. Lateral diffusion and reorientational time correlations of lipids close to the helix were slower than for the bulk ones, but not more than two or three times. Thus, we did not find any evidence of bound or frozen boundary lipids.     

Coarse-grained molecular dynamics simulations of membrane proteins and peptides

Molecular dynamics (MD) simulations provide a valuable approach to the dynamics, structure, and stability of membrane-protein systems. Coarse-grained (CG) models, in which small groups of atoms are treated as single particles, enable extended (>100 ns) timescales to be addressed. In this study, we explore how CG-MD methods that have been developed for detergents and lipids may be extended to membrane proteins. In particular, CG-MD simulations of a number of membrane peptides and proteins are used to characterize their interactions with lipid bilayers. CG-MD is used to simulate the insertion of synthetic model membrane peptides (WALPs and LS3) into a lipid (PC) bilayer. WALP peptides insert in a transmembrane orientation, whilst the LS3 peptide adopts an interfacial location, both in agreement with experimental biophysical data. This approach is extended to a transmembrane fragment of the Vpu protein from HIV-1, and to the coat protein from fd phage. Again, simulated protein/membrane interactions are in good agreement with solid state NMR data for these proteins. CG-MD has also been applied to an M3-M4 fragment from the CFTR protein. Simulations of CFTR M3-M4 in a detergent micelle reveal formation of an alpha-helical hairpin, consistent with a variety of biophysical data. In an I231D mutant, the M3-M4 hairpin is additionally stabilized via an inter-helix Q207/D231 interaction. Finally, CG-MD simulations are extended to a more complex membrane protein, the bacterial sugar transporter LacY. Comparison of a 200 ns CG-MD simulation of LacY in a DPPC bilayer with a 50 ns atomistic simulation of the same protein in a DMPC bilayer shows that the two methods yield comparable predictions of lipid-protein interactions. Taken together, these results demonstrate the utility of CG-MD simulations for studies of membrane/protein interactions.     

Implicit membrane treatment of buried charged groups: Application to peptide translocation across lipid bilayers

The energetic cost of burying charged groups in the hydrophobic core of lipid bilayers has been controversial, with simulations giving higher estimates than certain experiments. Implicit membrane approaches are usually deemed too simplistic for this problem. Here we challenge this view. The free energy of transfer of amino acid side chains from water to the membrane center predicted by IMM1 is reasonably close to all-atom free energy calculations. The shape of the free energy profile, however, for the charged side chains needs to be modified to reflect the all-atom simulation findings (IMM1-LF). Membrane thinning is treated by combining simulations at different membrane widths with an estimate of membrane deformation free energy from elasticity theory. This approach is first tested on the voltage sensor and the isolated S4 helix of potassium channels. The voltage sensor is stably inserted in a transmembrane orientation for both the original and the modified model. The transmembrane orientation of the isolated S4 helix is unstable in the original model, but a stable local minimum in IMM1-LF, slightly higher in energy than the interfacial orientation. Peptide translocation is addressed by mapping the effective energy of the peptide as a function of vertical position and tilt angle, which allows identification of minimum energy pathways and transition states. The barriers computed for the S4 helix and other experimentally studied peptides are low enough for an observable rate. Thus, computational results and experimental studies on the membrane burial of peptide charged groups appear to be consistent. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Molecular packing in 1-hexanol-DMPC bilayers studied by molecular dynamics simulation

The structure and molecular packing density of a "mismatched" solute, 1-hexanol, in lipid membranes of dimyristoyl phosphatidylcholine (DMPC) was studied by molecular dynamics simulations. We found that the average location and orientation of the hexanol molecules matched earlier experimental data on comparable systems. The local density or molecular packing in DMPC-hexanol was elucidated through the average Voronoi volumes of all heavy (non-hydrogen) atoms. Analogous analysis was conducted on trajectories from simulations of pure 1-hexanol and pure (hydrated) DMPC bilayers. The results suggested a positive volume change, DeltaV(m), of 4 cm(3) mol(-1) hexanol partitioned at 310 K in good accordance with experimental values. Analysis of the apparent volumes of each component in the pure and mixed states further showed that DeltaV(m) reflects a balance between a substantial increase in the packing density of the alcohol upon partitioning and an even stronger loosening in the packing of the lipid. Furthermore, analysis of Voronoi volumes along the membrane normal identifies a distinctive depth dependence of the changes in molecular packing. The outer (interfacial) part of the lipid acyl chains (up to C8) is stretched by about 4%. Concomitantly, the average lateral area per chain decreases and these two effects compensate so that the overall packing density in the outer region, where the hexanol molecules are located, remains practically constant. The core of the bilayer (C9-C13) is slightly thinned. The average lateral area per chain in this region expands, resulting in a looser packing density. The net effect in the core is a 2-3% decrease in density corresponding to a total volume increase of approximately 14 cm(3) mol(-1) hexanol partitioned.     

Electric field effects on membranes: gramicidin A as a test ground

Electric fields due to transmembrane potential differences or ionic gradients across the membrane are presumably crucial for many reactions across membranes or close to membranes like signal transduction, control of ion channels or the generation of neural impulses. Molecular dynamics simulations have been used to study the influence of external electric fields on a mixed gramicidin/phospholipid bilayer system. At high field strengths, formation of membrane electropores occurred both close and distal to the gramicidin. Gramicidin was found to stabilize the membrane adjacent to the protein but also at larger distances of up to 2-3 nm. As a result, membrane pore formation was found to be significantly suppressed for the mixed gramicidin/DMPC system. Moderate field strengths only weakly affected the structure and dynamics of the gramicidin. Spontaneous potassium passage events in external electric fields were observed for both the head-to-head helical conformation as well as for the double helical conformation of gramicidin A. The double-helical conformation was found to facilitate ion passage compared to the head-to-head helical dimer.     

Structural stability of myoglobin and glycomyoglobin: a comparative molecular dynamics simulation study

Glycoproteins are formed as the result of enzymatic glycosylation or chemical glycation in the body, and produced in vitro in industrial processes. The covalently attached carbohydrate molecule(s) confer new properties to the protein, including modified stability. In the present study, the structural stability of a glycoprotein form of myoglobin, bearing a glucose unit in the N-terminus, has been compared with its native form by the use of molecular dynamics simulation. Both structures were subjected to temperatures of 300 and 500 K in an aqueous environment for 10 ns. Changes in secondary structures and RMSD were then assessed. An overall higher stability was detected for glycomyoglobin, for which the most stable segments/residues were highlighted and compared with the native form. The simple addition of a covalently bound glucose is suggested to exert its stabilizing effect via increased contacts with surrounding water molecules, as well as a different pattern of interactions with neighbor residues.

Electronic supplementary material

The online version of this article (doi:10.1007/s10867-015-9383-2) contains supplementary material, which is available to authorized users.     

Detailed molecular dynamics simulations of model biological membranes containing cholesterol

Detailed molecular dynamics simulations performed to study the nature of lipid raft domains that appear in model membranes are reviewed in this paper. The described simulations were performed on hydrated bilayers containing binary mixtures of cholesterol with phospholipids and also on ternary mixtures containing cholesterol, a phospholipid with a high main transition temperature T_m, and a phospholipid with a low transition temperature T_m. These simulations provide qualitative and semi-quantitative information about cholesterol-lipid interactions and also a testing ground for major assumptions made to explain the nature of lipid rafts in model membranes.