Effects of deformability and thermal motion of lipid membrane on electroporation: by molecular dynamics simulations

Effects of mechanical properties and thermal motion of POPE lipid membrane on electroporation were studied by molecular dynamics simulations. Among simulations in which specific atoms of lipids were artificially constrained at their equilibrium positions using a spring with force constant of 2.0 kcal/(mol Ų) in the external electric field of 1.4 kcal/(mol Å e), only constraint on lateral motions of lipid tails prohibited electroporation while non-tail parts had little effects. When force constant decreased to 0.2 kcal/(mol Ų) in the position constraints on lipid tails in the external electric field of 2.0 kcal/(mol Å e), water molecules began to enter the membrane. Position constraints of lipid tails allow water to penetrate from both sides of membrane. Thermal motion of lipids can induce initial defects in the hydrophobic core of membrane, which are favorable nucleation sites for electroporation. Simulations at different temperatures revealed that as the temperature increases, the time taken to the initial pore formation will decrease.     

The influence of cholesterol on membrane protein structure,function, and dynamics studied by molecular dynamics simulations

The plasma membrane, which encapsulates human cells, is composed of a complex mixture of lipids and embedded proteins. Emerging knowledge points towards the lipids as having a regulating role in protein function. Furthermore, insight from protein crystallography has revealed several different types of lipids intimately bound to membrane proteins and peptides, hereby possibly pointing to a site of action for the observed regulation. Cholesterol is among the lipid membrane constituents most often observed to be co-crystallized with membrane proteins, and the cholesterol levels in cell membranes have been found to play an essential role in health and disease. Remarkably little is known about the mechanism of lipid regulation of membrane protein function in health as well as in disease. Herein, we review molecular dynamics simulation studies aimed at investigating the effect of cholesterol on membrane protein and peptide properties. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Proton transport across transient single-file water pores in a lipid membrane studied by molecular dynamics simulations.

To test the hypothesis that water pores in a lipid membrane mediate the proton transport, molecular dynamic simulations of a phospholipid membrane, in which the formation of a water pore is induced, are reported. The probability density of such a pore in the membrane was obtained from the free energy of formation of the pore, which was computed from the average force needed to constrain the pore in the membrane. It was found that the free energy of a single file of water molecules spanning the bilayer is 108(+/-10) kJ/mol. From unconstrained molecular dynamic simulations it was further deduced that the nature of the pore is very transient, with a mean lifetime of a few picoseconds. The orientations of water molecules within the pore were also studied, and the spontaneous translocation of a turning defect was observed. The combined data allowed a permeability coefficient for proton permeation across the membrane to be computed, assuming that a suitable orientation of the water molecules in the pore allows protons to permeate the membrane relatively fast by means of a wirelike conductance mechanism. The computed value fits the experimental data only if it is assumed that the entry of the proton into the pore is not rate limiting.     

Flexibility of ras lipid modifications studied by 2H solid-state NMR and molecular dynamics simulations

Human posttranslationally modified N-ras oncogenes are known to be implicated in numerous human cancers. Here, we applied a combination of experimental and computational techniques to determine structural and dynamical details of the lipid chain modifications of an N-ras heptapeptide in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Experimentally, 2H NMR spectroscopy was used to study oriented membranes that incorporated ras heptapeptides with two covalently attached perdeuterated hexadecyl chains. Atomistic molecular dynamics simulations of the same system were carried out over 100 ns including 60 DMPC and 4 ras molecules. Several structural and dynamical experimental parameters could be directly compared to the simulation. Experimental and simulated 2H NMR order parameters for the methylene groups of the ras lipid chains exhibited a systematic difference attributable to the absence of collective motions in the simulation and to geometrical effects. In contrast, experimental 2H NMR spin-lattice relaxation rates for Zeeman order were well reproduced in the simulation. The lack of slower collective motions in the simulation did not appreciably influence the relaxation rates at a Larmor frequency of 115.1 MHz. The experimental angular dependence of the 2H NMR relaxation rates with respect to the external magnetic field was also relatively well simulated. These relaxation rates showed a weak angular dependence, suggesting that the lipid modifications of ras are very flexible and highly mobile in agreement with the low order parameters. To quantify these results, the angular dependence of the 2H relaxation rates was calculated by an analytical model considering both molecular and collective motions. Peptide dynamics in the membrane could be modeled by an anisotropic diffusion tensor with principal values of Dparallel=2.1x10(9) s(-1) and Dperpendicular=4.5x10(5) s(-1). A viscoelastic fitting parameter describing the membrane elasticity, viscosity, and temperature was found to be relatively similar for the ras peptide and the DMPC host matrix. Large motional amplitudes and relatively short correlation times facilitate mixing and dispersal with the lipid bilayer matrix, with implications for the role of the full-length ras protein in signal transduction and oncogenesis.     

Insertion of Dengue E into lipid bilayers studied by neutron reflectivity and molecular dynamics simulations

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that the tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.     

Coarse-grained molecular dynamics simulations of the energetics of helix insertion into a lipid bilayer

Experimental and computational studies have indicated that hydrophobicity plays a key role in driving the insertion of transmembrane alpha-helices into lipid bilayers. Molecular dynamics simulations allow exploration of the nature of the interactions of transmembrane alpha-helices with their lipid bilayer environment. In particular, coarse-grained simulations have considerable potential for studying many aspects of membrane proteins, ranging from their self-assembly to the relation between their structure and function. However, there is a need to evaluate the accuracy of coarse-grained estimates of the energetics of transmembrane helix insertion. Here, three levels of complexity of model system have been explored to enable such an evaluation. First, calculated free energies of partitioning of amino acid side chains between water and alkane yielded an excellent correlation with experiment. Second, free energy profiles for transfer of amino acid side chains along the normal to a phosphatidylcholine bilayer were in good agreement with experimental and atomistic simulation studies. Third, estimation of the free energy profile for transfer of an arginine residue, embedded within a hydrophobic alpha-helix, to the center of a lipid bilayer gave a barrier of approximately 15 kT. Hence, there is a substantial barrier to membrane insertion for charged amino acids, but the coarse-grained model still underestimates the corresponding free energy estimate (approximately 29 kT) from atomistic simulations (Dorairaj, S., and Allen, T. W. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 4943-4948). Coarse-grained simulations were then used to predict the free energy profile for transfer of a simple model transmembrane alpha-helix (WALP23) across a lipid bilayer. The results indicated that a transmembrane orientation was favored by about -70 kT.     

Aggregation of small peptides studied by molecular dynamics simulations

Peptides and proteins tend to aggregate under appropriate conditions. The amyloid fibrils that are ubiquitously found among these structures are associated with major human diseases like Alzheimer's disease, type II diabetes, and various prion diseases. Lately, it has been observed that even very short peptides like tetra and pentapeptides can form ordered amyloid structures. Here, we present aggregation studies of three such small polypeptide systems, namely, the two amyloidogenic peptides DFNKF and FF, and a control (nonamyloidogenic) one, the AGAIL. The respective aggregation process is studied by all-atom Molecular Dynamics simulations, which allow to shed light on the fine details of the association and aggregation process. Our analysis suggests that naturally aggregating systems exhibit significantly diverse overall cluster shape properties and specific intermolecular interactions. Additional analysis was also performed on the previously studied NFGAIL system.     

Virus capsid dissolution studied by microsecond molecular dynamics simulations

Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.     

Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations

Ribonucleotide reductase (RNR) is necessary for production of the precursor deoxyribonucleotides for DNA synthesis. Class Ia RNR functions via a stable free radical in one of the two components protein R2. The enzyme mechanism involves long range (proton coupled) electron transfer between protein R1 and the tyrosyl radical in protein R2. Earlier experimental studies showed that p-alkoxyphenols inhibit RNR. Here, molecular docking and molecular dynamics simulations involving protein R2 suggest an inhibition mechanism for p-alkoxyphenols . A low energy binding pocket is identified in protein R2. The preferred configuration provides a structural basis explaining their specific binding to the Escherichia coli and mouse R2 proteins. Trp48 (E. coli numbering), on the electron transfer pathway, is involved in the interactions with the inhibitors. The relative order of the binding energies calculated for the phenol derivatives to protein R2 is correlated with earlier experimental data on inhibition efficiency, in turn related to increasing size of the hydrophobic alkyl substituents. Using the configuration identified by molecular docking as a starting point for molecular dynamics simulations, we find that the p-allyloxyphenol interrupts the catalytic electron transfer pathway of the R2 protein by forming hydrogen bonds with Trp48 and Asp237, thus explaining the inhibitory activity of p-alkoxyphenols.     

Structural determinants of MscL gating studied by molecular dynamics simulations

The mechanosensitive channel of large conductance (MscL) in prokaryotes plays a crucial role in exocytosis as well as in the response to osmotic downshock. The channel can be gated by tension in the membrane bilayer. The determination of functionally important residues in MscL, patch-clamp studies of pressure-conductance relationships, and the recently elucidated crystal structure of MscL from Mycobacterium tuberculosis have guided the search for the mechanism of MscL gating. Here, we present a molecular dynamics study of the MscL protein embedded in a fully hydrated POPC bilayer. Simulations totaling 3 ns in length were carried out under conditions of constant temperature and pressure using periodic boundary conditions and full electrostatics. The protein remained in the closed state corresponding to the crystal structure, as evidenced by its impermeability to water. Analysis of equilibrium fluctuations showed that the protein was least mobile in the narrowest part of the channel. The gating process was investigated through simulations of the bare protein under conditions of constant surface tension. Under a range of conditions, the transmembrane helices flattened as the pore widened. Implications for the gating mechanism in light of these and experimental results are discussed.     

DNA condensation by TmHU studied by optical tweezers,AFM and molecular dynamics simulations

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.     

Fast lipid disorientation at the onset of membrane fusion revealed by molecular dynamics simulations.

Membrane fusion is a key event in vesicular trafficking in every cell, and many fusion-related proteins have been identified. However, how the actual fusion event occurs has not been elucidated. By using molecular dynamics simulations we found that when even a small region of two membranes is closely apposed such that only a limited number of water molecules remain in the apposed area (e.g., by a fusogenic protein and thermal membrane fluctuations), dramatic lipid disorientation results within 100 ps-2 ns, which might initiate membrane fusion. Up to 12% of phospholipid molecules in the apposing layers had their alkyl chains outside the hydrophobic region, lying almost parallel to the membrane surface or protruding out of the bilayer by 2 ns after two membranes were closely apposed.     

CGDB: a database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

Structure, dynamics, and elasticity of free 16s rRNA helix 44 studied by molecular dynamics simulations

Molecular dynamics (MD) simulations were employed to investigate the structure, dynamics, and local base-pair step deformability of the free 16S ribosomal helix 44 from Thermus thermophilus and of a canonical A-RNA double helix. While helix 44 is bent in the crystal structure of the small ribosomal subunit, the simulated helix 44 is intrinsically straight. It shows, however, substantial instantaneous bends that are isotropic. The spontaneous motions seen in simulations achieve large degrees of bending seen in the X-ray structure and would be entirely sufficient to allow the dynamics of the upper part of helix 44 evidenced by cryo-electron microscopic studies. Analysis of local base-pair step deformability reveals a patch of flexible steps in the upper part of helix 44 and in the area proximal to the bulge bases, suggesting that the upper part of helix 44 has enhanced flexibility. The simulations identify two conformational substates of the second bulge area (bottom part of the helix) with distinct base pairing. In agreement with nuclear magnetic resonance (NMR) and X-ray studies, a flipped out conformational substate of conserved 1492A is seen in the first bulge area. Molecular dynamics (MD) simulations reveal a number of reversible alpha-gamma backbone flips that correspond to transitions between two known A-RNA backbone families. The flipped substates do not cumulate along the trajectory and lead to a modest transient reduction of helical twist with no significant influence on the overall geometry of the duplexes. Despite their considerable flexibility, the simulated structures are very stable with no indication of substantial force field inaccuracies.     

Sequence-dependent DNA deformability studied using molecular dynamics simulations

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein–DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein–DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.     

Distribution and favorable binding sites of pyrroloquinoline and its analogues in a lipid bilayer studied by molecular dynamics simulations

The distribution of 1H-pyrrolo[3,2-h]quinoline (PQ), 11H-dipyrido[2,3-a]carbazole (PC) and 7-azaindole (7AI) at a water/membrane interface has been investigated by molecular dynamics (MD) simulations. The MD study focused on favorable binding sites of the azaaromatic probes across a dipalmitoylphosphatidylcholine (DPPC) bilayer. Our simulations show that PQ and PC are preferably accommodated at the hydrocarbon core of the bilayer below the glycerol moiety. In addition, it is found that the hydrophobic aromatic parts of the probes are located inside a more ordered region of DPPC, consisting of hydrophobic lipid chains. In contrast to PQ and PC, 7AI is characterized by a broad distribution between a DPPC interface and water, so that the three preferable binding sites are found across a water/membrane interface. It is found that in the sequence 7AI-PQ-PC, due to the increase of the number of aromatic rings and, hence, the hydrophobic character of the probes, the depth of the probe localization is gradually shifted deeper inside the hydrocarbon core of the bilayer. We found that the probe-lipid hydrogen-bonding contributes weakly to the favorable localizations of the azaaromatic probes inside the DPPC bilayer, so that the probe localization is mainly driven by electrostatic dipole-dipole and van der Waals interactions.     

Efficiency of the monofunctionalized C60 fullerenes as membrane targeting agents studied by all-atom molecular dynamics simulations

Abstract

Transmembrane translocation of C₆₀ fullerenes functionalized by the single amino-derivative in neutral and charged forms was studies by extensive all-atom molecular dynamics simulations. It is shown that these complexes exhibit very strong affinity to the membrane core, but their spontaneous translocation through the membrane is not possible at practical time scale. In contrast, free amino derivatives translocate through the membrane much easier than their complexes with fullerenes, but do not have pronounced affinity to the membrane interior. Our results suggest that monofunctionalized C₆₀ could be extremely efficient membrane targeting agents, which facilitate accumulation of the water-soluble compounds in the hydrophobic core of lipid bilayer.     

Mechanically induced titin kinase activation studied by force-probe molecular dynamics simulations

The conversion of mechanical stress into a biochemical signal in a muscle cell requires a force sensor. Titin kinase, the catalytic domain of the elastic muscle protein titin, has been suggested as a candidate. Its activation requires major conformational changes resulting in the exposure of its active site. Here, force-probe molecular dynamics simulations were used to obtain insight into the tension-induced activation mechanism. We find evidence for a sequential mechanically induced opening of the catalytic site without complete domain unfolding. Our results suggest the rupture of two terminal beta-sheets as the primary unfolding steps. The low force resistance of the C-terminal relative to the N-terminal beta-sheet is attributed to their different geometry. A subsequent rearrangement of the autoinhibitory tail is seen to lead to the exposure of the active site, as is required for titin kinase activity. These results support the hypothesis of titin kinase as a force sensor.