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.     

Conducting-state properties of the KcsA potassium channel from molecular and Brownian dynamics simulations.

The mechanisms underlying transport of ions across the potassium channel are examined using electrostatic calculations and three-dimensional Brownian dynamics simulations. We first build open-state configurations of the channel with molecular dynamics simulations, by pulling the transmembrane helices outward until the channel attains the desired interior radius. To gain insights into ion permeation, we construct potential energy profiles experienced by an ion traversing the channel in the presence of other resident ions. These profiles reveal that in the absence of an applied field the channel accommodates three potassium ions in a stable equilibrium, two in the selectivity filter and one in the central cavity. In the presence of a driving potential, this three-ion state becomes unstable, and ion permeation across the channel is observed. These qualitative explanations are confirmed by the results of three-dimensional Brownian dynamics simulations. We find that the channel conducts when the ionizable residues near the extracellular entrance are fully charged and those near the intracellular side are partially charged. The conductance increases steeply as the radius of the intracellular mouth of the channel is increased from 2 A to 5 A. Our simulation results reproduce several experimental observations, including the current-voltage curves, conductance-concentration relationships, and outward rectification of currents.     

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.     

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.     

Orientation and conformation of a lipase at an interface studied by molecular dynamics simulations

Electron density profiles calculated from molecular dynamics trajectories are used to deduce the orientation and conformation of Thermomyces lanuginosa lipase and a mutant adsorbed at an air-water interface. It is demonstrated that the profiles display distinct fine structures, which uniquely characterize enzyme orientation and conformation. The density profiles are, on the nanosecond timescale, determined by the average enzyme conformation. We outline a computational scheme that from a single molecular dynamics trajectory allows for extraction of electron density profiles referring to different orientations of the lipase relative to an implicit interface. Profiles calculated for the inactive and active conformations of the lipase are compared with experimental electron density profiles measured by x-ray reflectivity for the lipase adsorbed at an air-water interface. The experimental profiles contain less fine structural information than the calculated profiles because the resolution of the experiment is limited by the intrinsic surface roughness of water. Least squares fits of the calculated profiles to the experimental profiles provide areas per adsorbed enzyme and suggest that Thermomyces lanuginosa lipase adsorbs to the air-water interface in a semiopen conformation with the lid oriented away from the interface.     

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.     

An alamethicin channel in a lipid bilayer: molecular dynamics simulations

We present the results of 2-ns molecular dynamics (MD) simulations of a hexameric bundle of Alm helices in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer. These simulations explore the dynamic properties of a model of a helix bundle channel in a complete phospholipid bilayer in an aqueous environment. We explore the stability and conformational dynamics of the bundle in a phospholipid bilayer. We also investigate the effect on bundle stability of the ionization state of the ring of Glu18 side chains. If all of the Glu18 side chains are ionised, the bundle is unstable; if none of the Glu18 side chains are ionized, the bundle is stable. pKA calculations suggest that either zero or one ionized Glu18 is present at neutral pH, correlating with the stable form of the helix bundle. The structural and dynamic properties of water in this model channel were examined. As in earlier in vacuo simulations (Breed et al., 1996 .Biophys. J. 70:1643-1661), the dipole moments of water molecules within the pore were aligned antiparallel to the helix dipoles. This contributes to the stability of the helix bundle.     

Ion solvation and structural stability in a sodium channel investigated by molecular dynamics calculations

The stability and ion binding properties of the homo-tetrameric pore domain of a prokaryotic, voltage-gated sodium channel are studied by extensive all-atom molecular dynamics simulations, with the channel protein being embedded in a fully hydrated lipid bilayer. It is found that Na(+) ion presents in a mostly hydrated state inside the wide pore of the selectivity filter of the sodium channel, in sharp contrast to the nearly fully dehydrated state for K(+) ions in potassium channels. Our results also indicate that Na(+) ions make contact with only one or two out of the four polypeptide chains forming the selectivity filter, and surprisingly, the selectivity filter exhibits robust stability for various initial ion configurations even in the absence of ions. These findings are quite different from those in potassium channels. Furthermore, an electric field above 0.5V/nm is suggested to be able to induce Na(+) permeation through the selectivity filter.     

DNA base pair hybridization and water-mediated metastable structures studied by molecular dynamics simulations

The base pair hybridization of a DNA segment was studied using molecular dynamics simulation. The results show the obvious correlation between the probability of successful hybridization and the accessible surface area to water of two successive base pairs, including the unpaired base pair adjacent to paired base pair and this adjacent paired base pair. Importantly, two metastable structures in an A-T base pair were discovered by the analysis of the free energy landscape. Both structures involved addition of a water molecule to the linkage between the two nucleobases in one base pair. The existence of the metastable structures provide potential barriers to the Watson-Crick base pair, and numerical simulations show that those potential barriers can be surmounted by thermal fluctuations at higher temperatures. These studies contribute an important step toward the understanding of the mechanism in DNA hybridization, particularly the effect of temperature on DNA hybridization and polymerase chain reaction. These observations are expected to be helpful for facilitating experimental bio/nanotechnology designs involving fast hybridization.     

Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations

Folding reaction of goat alpha-lactalbumin has been studied by stopped-flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the alpha-domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native-state stability of goat alpha-lactalbumin against the guanidine hydrochloride-induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild-type and mutant goat alpha-lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high-temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter-residue contacts and with the water molecule penetration in the 400-K simulation as well as in four of the six 600-K simulations. Disruption of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report.     

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.     

CO migration pathways in cytochrome P450cam studied by molecular dynamics simulations

Previous laser flash photolysis investigations between 100 and 300 K have shown that the kinetics of CO rebinding with cytochrome P450(cam)(camphor) consist of up to four different processes revealing a complex internal dynamics after ligand dissociation. In the present work, molecular dynamics simulations were undertaken on the ternary complex P450(cam)(cam)(CO) to explore the CO migration pathways, monitor the internal cavities of the protein, and localize the CO docking sites. One trajectory of 1 nsec with the protein in a water box and 36 trajectories of 1 nsec in the vacuum were calculated. In each trajectory, the protein contained only one CO ligand on which no constraints were applied. The simulations were performed at 200, 300, and 320 K. The results indicate the presence of seven CO docking sites, mainly hydrophobic, located in the same moiety of the protein. Two of them coincide with xenon binding sites identified by crystallography. The protein matrix exhibits eight persistent internal cavities, four of which corresponding to the ligand docking sites. In addition, it was observed that water molecules entering the protein were mainly attracted into the polar pockets, far away from the CO docking sites. Finally, the identified CO migration pathways provide a consistent interpretation of the experimental rebinding kinetics.     

Structure and dynamics of the full-length M1 muscarinic acetylcholine receptor studied by molecular dynamics simulations

The three-dimensional structure of full-length structure of the M₁ muscarinic receptor was obtained through the fragmental homology modeling procedure. A 10-ns molecular dynamics (MD) simulation of the protein imbedded in a lipid slab and surrounded by water molecules was further used to relax the model. It was found that the homology model corresponded to the conformation in the ground state, since no significant motions of the backbone of transmembrane domains were observed. Furthermore, the reliability of the model was validated by analyzing key inter-helical contacts, sidechain-sidechain interactions, the formation of stable aromatic microdomains (clusters) and the docking of acetylcholine to its binding site. Moreover, a few conserved interactions observed in the X-ray structure of rhodopsin, such as inter-helical sidechain-sidechain hydrogen bonds were accurately reproduced in the MD simulation. The coupling of ACh to its binding site was found to be dominated by π-cation and salt bridge interactions, while its conformational space was restrained through van der Waals and hydrogen bond interactions. In general, such features were in very good agreement with the available experimental as well as with theoretical data. Considering the above, the structural information obtained in this study can be used a starting point to investigate the activation mechanism of the receptor and the ability to develop selective agonists and allosteric modulators which could be used for the treatment of Alzheimer’s disease.     

Interaction of anesthetics with open and closed conformations of a potassium channel studied via molecular dynamics and normal mode analysis

A variety of experiments suggest that membrane proteins are important targets of anesthetic molecules, and that ion channels interact differently with anesthetics in their open and closed conformations. The availability of an open and a closed structural model for the KirBac1.1 potassium channel has made it possible to perform a comparative analysis of the interactions of anesthetics with the same channel in its open and closed states. To this end, all-atom molecular dynamics simulations supplemented by normal mode analysis have been employed to probe the interactions of the inhalational anesthetic halothane with both an open and closed conformer of KirBac1.1 embedded in a lipid bilayer. Normal mode analysis on the closed and open channel, in the presence and absence of halothane, reveals that the anesthetic modulates the global as well as the local dynamics of both conformations differently. In the case of the open channel, the observed reduction of flexibility of residues in the inner helices suggests a functional modification action of anesthetics on ion channels. In this context, preferential quenching of the aromatic residue motion and modulation of global dynamics by halothane may be seen as steps toward potentiating or favoring open state conformations. These molecular dynamics simulations provide the first insights into possible specific interactions between anesthetic molecules and ion channels in different conformations.     

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.     

Interactions of liquid crystal-forming molecules with phospholipid bilayers studied by molecular dynamics simulations

Recent experiments have shown that liquid crystals can be used to image mammalian cell membranes and to amplify structural reorganization in phospholipid-laden liquid crystal-aqueous interfaces. In this work, molecular dynamics simulations were employed to explore the interactions between commonly used liquid crystal-forming molecules and phospholipid bilayers. In particular, umbrella sampling was used to obtain the potential of mean force of 4-cyano-4'-pentylbiphenyl (5CB) and 4'-(3,4-difluor-phenyl)-4-pentyl-bicylohexyl (5CF) molecules partitioning into a dipalmitoylphosphatidylcholine bilayer. In addition, results of simulations are presented for systems consisting of a fully hydrated bilayer with 5CB or 5CF molecules at the lowest (4.5 mol %) and highest (20 mol %) concentrations used in recent laboratory experiments. It is found that mesogens preferentially partition from the aqueous phase into the membrane; the potential of mean force exhibits highly favorable free energy differences for partitioning (-18 k(B)T for 5CB and -26 k(B)T for 5CF). The location and orientation of mesogens associated with the most stable free energies in umbrella sampling simulations of dilute systems were found to be consistent with those observed in liquid-crystal-rich bilayers. It is found that the presence of mesogens in the bilayer enhances the order of lipid acyl tails, and changes the spatial and orientational arrangement of lipid headgroup atoms. These effects are more pronounced at higher liquid-crystal concentrations. In comparing the behavior of 5CB and 5CF, a stronger spatial correlation (i.e., possibly leading to aggregation) is observed between 5CB molecules within a bilayer than between 5CF molecules. Also, the range of molecular orientations and positions along the bilayer normal is larger for 5CB molecules. At the same time, 5CF molecules were found to bind more strongly to lipid headgroups, thereby slowing the lateral motion of lipid molecules.