Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the “boson peak” observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure. Received: 12 October 1998 / Revised version: 19 February 1999 / Accepted: 1 March 1999     

Long-term molecular dynamics simulation of copper plastocyanin in water

A long molecular dynamics simulation (1.1 ns) of fully hydrated plastocyanin has been performed and analysed to relate protein dynamics to structural elements and functional properties. The solvated structure is described in detail by the analysis of H-bond network. During all the simulation, the crystal H-bond network is maintained in the beta-sheet regions, while several H-bonds are broken or formed on the external surface of the protein. To evaluate whether such changes could be due to conformational rearrangements or to solvent competition, we have examined the average number of H-bonds between protein atoms and water molecules, and the root mean square deviations from crystal structure as a function of protein residues. Protein mobility and flexibility have been examined by positional and dihedral angle rms fluctuations. Finally, cross-correlation maps have revealed the existence of correlated motions among residues connected by hydrogen bonds.     

Active site modeling in copper azurin molecular dynamics simulations

Active site modeling in molecular dynamics simulations is investigated for the reduced state of copper azurin. Five simulation runs (5 ns each) were performed at room temperature to study the consequences of a mixed electrostatic/constrained modeling for the coordination between the metal and the polypeptide chain, using for the ligand residues a set of charges that is modified with respect to the apo form of the protein by the presence of the copper ion.The results show that the different charge values do not lead to relevant effects on the geometry of the active site of the protein, as long as bond distance constraints are used for all the five ligand atoms. The distance constraint on the O atom of Gly45 can be removed without altering the active site geometry. The coordination between Cu and the other axial ligand Met121 is outlined as being flexible. Differences are found between the bonds of the copper ion with the two apparently equivalent N¹ atoms of His46 and His117.The overall findings are discussed in connection with the issue of determining a model for the active site of azurin suitable to be used in molecular dynamics simulations under unfolding conditions. Figure Model of azurin active site. Copper ligand residues are cut off at C position except Gly45, for which the portion of backbone connecting it to His46 is shown. Only polar H atoms are shown. All atoms are in standard colors (Cu in violet), and the five ligands are labeled     

Evidence of reduced flexibility in disulfide bridge-depleted azurin: a molecular dynamics simulation study.

Two molecular dynamics simulations have been performed for 2 ns, at room temperature, on fully hydrated wild type and Cys3Ala/Cys26Ala double-mutant azurin, to investigate the role of the unique disulfide bridge on the structure and dynamics of the protein. The results show that the removal of the [bond]SS[bond] bond does not affect the structural features of the protein, whereas alterations of the dynamical properties are observed. The root mean square fluctuations of the atomic positions are, on average, considerably reduced in the azurin mutant with respect to the wild type form. The number of intramolecular hydrogen bonds between protein backbone atoms that are lost during the simulation, with respect to the starting configuration, are reduced in the absence of the disulfide bond. The analysis of the dynamical cross-correlation map, characterising the protein co-ordinated internal motions, demonstrates in the mutated azurin a significant decrease in anti-correlated displacements between protein residues, with the only exception occurring in the region of the mutation sites. The overall findings show a relevant reduction in flexibility as a consequence of the disulfide bridge depletion in azurin, suggesting that the [bond]SS[bond] bond is a structural element which significantly contributes to the dynamic properties of the native protein.     

Concerted motions in copper plastocyanin and azurin: an essential dynamics study

Essential dynamics analysis of molecular dynamics simulation trajectories (1.1 ns) of two copper containing electron transfer proteins, plastocyanin and azurin, has been performed. The protein essential modes have been analysed in order to identify large concerted motions which could be relevant for the electron transfer function exerted by these proteins. The analysis, conducted for temporal windows of different lengths along the protein trajectories, shows a rapid convergence and indicates that for both the proteins the predominant internal motions occur in a subspace of only a few degrees of freedom. Moreover, it is found that for both the proteins the likely binding sites (i.e. the hydrophobic and negative patches) with the reaction partners move in a concerted fashion with a few structural regions far from the active site. Such results are discussed in connection with the possible involvement of large concerted motions in the recognition and binding interaction with physiological electron transfer partners.     

Protein structure and dynamics in nonaqueous solvents: insights from molecular dynamics simulation studies

Protein structure and dynamics in nonaqueous solvents are here investigated using molecular dynamics simulation studies, by considering two model proteins (ubiquitin and cutinase) in hexane, under varying hydration conditions. Ionization of the protein groups is treated assuming "pH memory," i.e., using the ionization states characteristic of aqueous solution. Neutralization of charged groups by counterions is done by considering a counterion for each charged group that cannot be made neutral by establishing a salt bridge with another charged group; this treatment is more physically reasonable for the nonaqueous situation, contrasting with the usual procedures. Our studies show that hydration has a profound effect on protein stability and flexibility in nonaqueous solvents. The structure becomes more nativelike with increasing values of hydration, up to a certain point, when further increases render it unstable and unfolding starts to occur. There is an optimal amount of water, approximately 10% (w/w), where the protein structure and flexibility are closer to the ones found in aqueous solution. This behavior can explain the experimentally known bell-shaped dependence of enzyme catalysis on hydration, and the molecular reasons for it are examined here. Water and counterions play a fundamental and dynamic role on protein stabilization, but they also seem to be important for protein unfolding at high percentages of bound water.     

Computational design,structure refinement and molecular dynamics simulation of novel engineered serratiopeptidase analogs

Communicated by Ramaswamy H. Sarma     

Modulation of the self-assembled structure of biomolecules: coarse grained molecular dynamics simulation

The mechanisms governing the self-assembled structure of biomolecules (single chain and bundle of chains) are studied with an AB copolymer model via the coarse grained molecular dynamics simulations. Non-local hydrophobic interaction is found to play a critical role in the pattern formation of the assembled structure of polymer chains. We show that the polymer structure could be controlled by adjusting the balance between local (short range) and non-local (long range) hydrophobic interaction which are influenced by various factors such as the sequences, chain length, stiffness, confinement, and the topology of polymers. In addition, the competition between the intrachain hydrophobic interaction and interchain hydrophobic interaction determines the structural transition of the chain bundles. This work may provide important insights into the fundamental physics in the structure control and the self-assembly of biomolecules for various practical applications.     

Crystal structure and molecular dynamics simulation of ubiquitin-like domain of murine parkin

Parkin is the gene product identified as the major cause of autosomal recessive juvenile Parkinsonism (AR-JP). Parkin, a ubiquitin ligase E3, contains a unique ubiquitin-like domain in its N-terminus designated Uld which is assumed to be a interaction domain with the Rpn 10 subunit of 26S proteasome. To elucidate the structural and functional role of Uld in parkin at the atomic level, the X-ray crystal structure of murine Uld was determined and a molecular dynamics simulation of wild Uld and its five mutants (K27N, R33Q, R42P, K48A and V56E) identified from AR-JP patients was performed. Murine Uld consists of two alpha helices [Ile23-Arg33 (alpha1) and Val56-Gln57 (alpha2)] and five beta strands [Met1-Phe7 (beta1), Tyr11-Asp18 (beta2), Leu41-Phe45 (beta3), Lys48-Pro51 (beta4) and Ser65-Arg72 (beta5)] and its overall structure is essentially the same as that of human ubiquitin with a 1.22 A rmsd for the backbone atoms of residues 1-76; however, the sequential identity and similarity between both molecules are 32% and 63%, respectively. This close resemblance is due to the core structure built by same hydrogen bond formations between and within the backbone chains of alpha1 and beta1/2/5 secondary structure elements and by nearly the same hydrophobic interactions formed between the nonpolar amino acids of their secondary structures. The side chain NetaH of Lys27 on the alpha1 helix was crucial to the stabilization of the spatial orientations of beta3 and beta4 strands, possible binding region with Rpn 10 subunit, through three hydrogen bonds. The MD simulations showed the K27N and R33Q mutations increase the structural fluctuation of these beta strands including the alpha1 helix. Reversely, the V56E mutant restricted the spatial flexibility at the periphery of the short alpha2 helix by the interactions between the polar atoms of Glu56 and Ser19 residues. However, a large fluctuation of beta4 strand with respect to beta5 strand was induced in the R42P mutant, because of the impossibility of forming paired hydrogen bonds of Pro for Arg42 in wild Uld. The X-ray structure showed that the side chains of Asp39, Gln40 and Arg42 at the N-terminal periphery of beta3 strand protrude from the molecular surface of Uld and participate in hydrogen bonds with the polar residues of neighboring Ulds. Thus, the MD simulation suggests that the mutation substitution of Pro for Arg42 not only causes the large fluctuation of beta3 strand in the Uld but also leads to the loss of the ability of Uld to trap the Rpn 10 subunit. In contrast, the MD simulation of K48A mutant showed little influence on the beta3-beta4 loop structure, but a large fluctuation of Lys48 side chain, suggesting the importance of flexibility of this side chain for the interaction with the Rpn 10 subunit. The present results would be important in elucidating the impaired proteasomal binding mechanism of parkin in AR-JP.     

Molecular dynamics simulation of solvated azurin: correlation between surface solvent accessibility and water residence times

A system containing the globular protein azurin and 3,658 water molecules has been simulated to investigate the influence on water dynamics exerted by a protein surface. Evaluation of water mean residence time for elements having different secondary structure did not show any correlation. Identically, comparison of solvent residence time for atoms having different charge and polarity did not show any clear trend. The main factor influencing water residence time in proximity to a specific site was found to be its solvent accessibility. In detail for atoms belonging to lateral chains and having solvent-accessible surface lower than approximately 16 A(2)a relation is found for which charged and polar atoms are surrounded by water molecules characterized by residence times longer than the non polar ones. The involvement of the low accessible protein atom in an intraprotein hydrogen bond further modulates the length of the water residence time. On the other hand for surfaces having high solvent accessibility, all atoms, independently of their character, are surrounded by water molecules which rapidly exchange with the bulk solvent. Proteins 2000;39:56-67.     

A molecular dynamics simulation study of the solvent isotope effect on copper plastocyanin

The effect of heavy water on the structure and dynamics of copper plastocyanin as well as on some aspects of the solvent dynamics at the protein-solvent interfacial region have been investigated by molecular dynamics simulation. The simulated system has been analyzed in terms of the atomic root mean square deviation and fluctuations, intraprotein H-bond pattern, dynamical cross-correlation map and the results have been compared with those previously obtained for plastocyanin in H2O (Ciocchetti et al. Biophys. Chem. 69 (1997), 185-198). The simulated plastocyanin structure in the two solvents, averaging 1 ns, is very similar along the beta-structure regions, while the most significant differences are registered, analogous to the turns and the regions likely involved in the electron transfer pathway. Moreover, plastocyanin in D2O shows an increase in the number of both the intraprotein H-bonds and the residues involved in correlated motions. An analysis of the protein-solvent coupling evidenced that D2O makes the H-bond formation more difficult with the solvent molecules for positively charged and polar residues, while an opposite trend is observed for negatively charged residues. On the other hand, the frequency of exchange of the solvent molecules involved in the protein-solvent H-bond formation is significantly depressed in D2O. The results are discussed also in connection with protein functionality and briefly with some experimental results connected with the thermostability of proteins in D2O.     

Transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations.

To probe the fundamentals of membrane/protein interactions, all-atom multi-nanosecond molecular dynamics simulations were conducted on a single transmembrane poly(32)alanine helix in a fully solvated dimyristoyphosphatidylcholine (DMPC) bilayer. The central 12 residues, which interact only with the lipid hydrocarbon chains, maintained a very stable helical structure. Helical regions extended beyond these central 12 residues, but interactions with the lipid fatty-acyl ester linkages, the lipid headgroups, and water molecules made the helix less stable in this region. The C and N termini, exposed largely to water, existed as random coils. As a whole, the helix tilted substantially, from perpendicular to the bilayer plane (0 degree) to a 30 degrees tilt. The helix experienced a bend at its middle, and the two halves of the helix at times assumed substantially different tilts. Frequent hydrogen bonding, of up to 0.7 ns in duration, occurred between peptide and lipid molecules. This resulted in correlated translational diffusion between the helix and a few lipid molecules. Because of the large variation in lipid conformation, the lipid environment of the peptide was not well defined in terms of "annular" lipids and on average consisted of 18 lipid molecules. When compared with a "neat" bilayer without peptide, no significant difference was seen in the bilayer thickness, lipid conformations or diffusion, or headgroup orientation. However, the lipid hydrocarbon chain order parameters showed a significant decrease in order, especially in those methylene groups closest to the headgroup.     

Solution structure of hyaluronic acid oligomers by experimental and theoretical NMR, and molecular dynamics simulation

The conformational properties of hyaluronic acid (HA) oligomers in aqueous solution were investigated by combining high-resolution NMR experimental results, theoretical simulation of NMR two-dimensional (2D) spectra by Complete Relaxation Matrix Analysis (CORMA), and molecular dynamics calculations. New experimental findings recorded for the tetra- and hexasaccharides enabled the stiffness of the HA and its viscoelastic properties to be interpreted. In particular, rotating frame nuclear Overhauser effect spectroscopy spectra provided new information about the arrangement of the glycosidic linkage. From (13)C NMR relaxation the rotational correlation time (tau(c)) were determined. The tau(c) were employed in the calculation of geometrical constraints, by using the MARDIGRAS algorithm. Restrained simulated annealing and 1 ns of unrestrained molecular dynamic simulations were performed on the hexasaccharide in a box of 1215 water molecules. The beta(1 --> 3) and beta(1 --> 4) glycosidic links were found to be rigid. The lack of rotational degree of freedom is due to direct and/or water-mediated interresidue hydrogen bonding. Both single or tandem water bridges were found between carboxylate group and N-acetil group. The carboxylate group of glucuronic acid is not involved in a direct link with the amide group of N-acetyl glucosamine and this facilitated bonding between the residue and the water molecules.     

Molecular dynamics simulation and steered molecular dynamics simulation on irisin dimers

Irisin is found closely associated with promoting the browning of beige fat cells in white adipose tissue. The crystal structure reveals that irisin forms a continuous inter-subunit β-sheet dimer. Here, molecular dynamics (MD) simulation and steered molecular dynamics (SMD) simulation were performed to investigate the dissociation process and the intricate interactions between the two irisin monomers. In the process of MD, the interactions between the monomers were roughly analyzed through the average numbers of both hydrophobic contacts and H-bonds. Then, SMD was performed to investigate the accurate interaction energy between the monomers. By the analysis of dissociation energy, the van der Waals (vdW) force was identified as the major energy to maintain the dimer structure, which also verified the results of MD simulation. Meanwhile, 11 essential residues were discovered by the magnitude of rupture force during dissociation. Among them, residues Arg75, Glu79, Ile77, Ala88, and Trp90 were reported in a previous study using the method of mutagenesis and size exclusion chromatography, and several new important residues (Arg72, Leu74, Phe76, Gln78, Val80, and Asp91) were also identified. Interestingly, the new important residues that we discovered and the important residues that were reported are located in the opposite side of the β-sheet of the dimer.     

Membrane electroporation: a molecular dynamics simulation

We present results of molecular dynamics simulations of lipid bilayers under a high transverse electrical field aimed at investigating their electroporation. Several systems are studied, namely 1), a bare bilayer, 2), a bilayer containing a peptide nanotube channel, and 3), a system with a peripheral DNA double strand. In all systems, the applied transmembrane electric fields (0.5 V.nm(-1) and 1.0 V.nm(-1)) induce an electroporation of the lipid bilayer manifested by the formation of water wires and water channels across the membrane. The internal structures of the peptide nanotube assembly and that of the DNA strand are hardly modified under field. For system 2, no perturbation of the membrane is witnessed at the vicinity of the channel, which indicates that the interactions of the peptide with the nearby lipids stabilize the bilayer. For system 3, the DNA strand migrates to the interior of the membrane only after electroporation. Interestingly enough, switching of the external transmembrane potential in cases 1 and 2 for few nanoseconds is enough to allow for complete resealing and reconstitution of the bilayer. We provide evidence that the electric field induces a significant lateral stress on the bilayer, manifested by surface tensions of magnitudes in the order of 1 mN.m(-1). This study is believed to capture the essence of several dynamical phenomena observed experimentally and provides a framework for further developments and for new applications.     

Oligoglycine surface structures: molecular dynamics simulation

The full-atomic molecular dynamics (MD) simulation of adsorption mode for diantennary oligoglycines [H-Gly4-NH(CH2)5]2 onto graphite and mica surface is described. The resulting structure of adsorption layers is analyzed. The peptide second structure motives have been studied by both STRIDE (structural identification) and DSSP (dictionary of secondary structure of proteins) methods. The obtained results confirm the possibility of polyglycine II (PGII) structure formation in diantennary oligoglycine (DAOG) monolayers deposited onto graphite surface, which was earlier estimated based on atomic-force microscopy measurements.     

Effects of single nucleotide polymorphisms on human N-acetyltransferase 2 structure and dynamics by molecular dynamics simulation

Background

Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear.

Methodology/Principal Findings

We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential.

Conclusions/Significance

Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms.     

Statistical prediction and molecular dynamics simulation

We describe a statistical approach to the validation and improvement of molecular dynamics simulations of macromolecules. We emphasize the use of molecular dynamics simulations to calculate thermodynamic quantities that may be compared to experimental measurements, and the use of a common set of energetic parameters across multiple distinct molecules. We briefly review relevant results from the theory of stochastic processes and discuss the monitoring of convergence to equilibrium, the obtaining of confidence intervals for summary statistics corresponding to measured quantities, and an approach to validation and improvement of simulations based on out-of-sample prediction. We apply these methods to replica exchange molecular dynamics simulations of a set of eight helical peptides under the AMBER potential using implicit solvent. We evaluate the ability of these simulations to quantitatively reproduce experimental helicity measurements obtained by circular dichroism. In addition, we introduce notions of statistical predictive estimation for force-field parameter refinement. We perform a sensitivity analysis to identify key parameters of the potential, and introduce Bayesian updating of these parameters. We demonstrate the effect of parameter updating applied to the internal dielectric constant parameter on the out-of-sample prediction accuracy as measured by cross-validation.