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R L Ornstein

Publications and source records attributed to R L Ornstein.

At least 19 recordsLinked to original sources
PubMed2001

Mobility of the active site bound paraoxon and sarin in zinc-phosphotriesterase by molecular dynamics simulation and quantum chemical calculation.

The kinetic data published on phosphotriesterase (PTE), with various complexed metals, clearly indicates that the P=O and P=S bonds of phosphotriester and thiophosphotriester substrates, respectively, are strongly polarized by one or both of the active site complexed metal ions. However, this observation is not consistent with the three-dimensional X-ray crystal structure of zinc-substituted PTE with active site bound substrate analogue diethyl 4-methylbenzylphosphonate. In this structure, the distance between the phosphoryl oxygen and the nearest zinc is 3.4 A, a distance too large to afford strong polarization. In the present paper, the geometry and mobility of various PTE active site-substrate complexes are examined by performing both molecular dynamics (MD) simulations and quantum mechanical calculations. Two known substrates are considered, paraoxon and sarin, although their turnover rates vary about 100-fold. The results indicate that PTE forms a complex with either substrate in which the phosphoryl oxygen becomes strongly coordinated with the less buried zinc atom. It is shown that the geometry of the active site is changed when the protein is immersed in a water bath and relaxed by MD. The most substantial conformational change is the opening of the gateway in a pocket where the location of the leaving group is expected. The opening is observed for the pure enzyme as well as for the enzyme/substrate complexes and it ranges from 11 to 18 A. It is also shown that the pockets, in which the substrate substituents are localized, exhibit different flexibility and interact with the substrate with coordinated conformational adjustments.

Aryldialkylphosphatase↗
PubMed1999

Molecular dynamics simulations of a protein-protein dimer: particle-mesh Ewald electrostatic model yields far superior results to standard cutoff model.

In this article we present two 1000 ps molecular dynamics simulations on the rat micro-glutathione S-transferase dimeric enzyme in complex with the product 1-(S-glutathionyl)-2,4-dinitrobenzene, in a periodic box with explicit solvent molecules, and investigate the effect of long-range electrostatics models on the structure and dynamics of the dimer and its components. One simulation used the standard cutoff method (10A), whilst the other used the particle-mesh Ewald (PME) method. We monitored the root mean-square atomic deviation (RMSD) from the initial crystal structure to examine the convergence of both simulations, as well as several other structural parameters such as the distance between active sites, rigid body rotation between domains in subunits, radius of gyration, B-factors, number of hydrogen bonds and salt bridges and solvent-accessible surface area. For example, with the PME method, the dimer structure remains much closer to the initial crystallographic structure with an average RMSD of 1.3A +/- 0.1A and 1.0A +/- 0.1A for all heavy and backbone atoms, respectively, in the last 200 ps; the respective values for the cutoff simulation are 4.7A +/- 0.3A and 4.2A +/- 0.3A. The large deviations observed in the cutoff simulation severely affected the stability of the enzyme dimer and its complex with the bound product. This finding is contrary to that found in a similar study of the monomeric protein ubiquitin [Fox, T. & Kollman, P. A. Proteins Struct. Func. Genet. 25, 315-334 (1996)]. Unlike the earlier published work, the present study provides evidence that the standard cutoff method is not generally valid for the study of protein complexes, or their subunits.

Animals↗
PubMed1998

Inherent DNA curvature and flexibility correlate with TATA box functionality.

Four 1.5 ns molecular dynamics (MD) simulations were performed on the d(GCTATAAAAGGG).d(CCCTTTTATAGC) double helix dodecamer bearing the Adenovirus major late promoter TATA element and three iso-composition mutants for which physical and biochemical data are available from the same laboratory. Three of these DNA sequences experimentally induce tight binding with the TATA box binding protein (TBP) and induce high transcription rates; the other DNA sequence induces much lower TBP binding and transcription. The x-ray crystal structures have previously shown that the duplex DNA in DNA-TBP complexes are highly bent. We performed and analyzed MD simulations for these four DNAs, whose experimental structures are not available, in order to address the issue of whether inherent DNA structure and flexibility play a role in establishing these observed preferences. A comparison of the experimental and simulated results demonstrated that DNA duplex sequence-dependent curvature and flexibility play a significant role in TBP recognition, binding, and transcriptional activation.

Adenoviridae↗
PubMed1997

Protein hinge bending as seen in molecular dynamics simulations of native and M61 mutant T4 lysozymes.

A dynamical model of interdomain "hinge bending" of T4 lysozyme in aqueous solution has been developed on the basis of molecular dynamics (MD) simulation. The MD model study provides a description of the conformational reorganization expected to occur for the protein in aqueous solution as compared to the crystalline environment. Three different 500 ps molecular dynamics simulations were calculated, each using a distinctly different crystal conformation of T4 lysozyme as the starting points of the MD simulations. Crystal structures of wild-type lysozyme and "open" and "closed" forms of M61 variant structures were analyzed in this study. Large-scale, molecular-conformational rearrangements were observed in all three simulations, and the largest structural change was found for the open form of the M61 allomorph. All three simulated proteins had closed relative to the wild-type crystal structure, and the closure of the "jaws" of the active site cleft occurred gradually over the time course of the trajectories. The time average MD structures, calculated over the final 50 ps of each trajectory, had all adapted to conformations more similar to each other than to their incipient crystal forms. Using a similar MD protocol on cytochrome P450BM-3 [M. D. Paulsen and R. L. Ornstein (1995) Proteins: Structure Function and Genetics, Vol. 27, pp. 237-243] we have found that the opposite type of motion relative to the starting crystal structure, that is, the open form of the crystal structure, had opened to a greater degree relative to the incipient crystal structure form. Therefore we do not believe that either result is merely a simulation artifact, but rather the protein dynamics are due to protein relaxation in the absence of crystal packing forces in the simulated solution environments.

Bacteriophage T4↗
PubMed1997

Molecular dynamics study of time-correlated protein domain motions and molecular flexibility: cytochrome P450BM-3.

Time-correlated atomic motions were used to characterize protein domain boundaries from atomic coordinates generated by molecular dynamics simulations. A novel application of the dynamical cross-correlation matrix (DCCM) analysis tool was used to help identify putative protein domains. In implementing this new approach, several DCCM maps were calculated, each using a different coordinate reference frame from which protein domain boundaries and protein domain residue constituents could be identified. Cytochrome P450BM-3, from Bacillus megaterium, was used as the model protein in this study. The analyses indicated that the simulated protein comprises three distinct domain regions; in contrast, only two protein domains were identified in the original crystal structure report. Specifically, the DCCM analyses showed that the F-G helix region was a separate domain entity and not a part of the alpha domain, as previously designated. The simulations demonstrated that the domain motions of the F-G helix region effected both the size and shape of the enzyme active site, and that the dynamics of the F-G helix domain could possibly control access of substrate to the binding pocket.

Bacillus megaterium↗
PubMed1997

Effect of periodic box size on aqueous molecular dynamics simulation of a DNA dodecamer with particle-mesh Ewald method.

The particle-mesh Ewald (PME) method is considered to be both efficient and accurate for the evaluation of long-range electrostatic interactions in large macromolecular systems being studied by molecular dynamics simulations. This method assumes "infinite" periodic boundary conditions resembling the symmetry of a crystal environment. Can such a "solid-state" method accurately portray a macromolecular solute such as DNA in solution? To address this issue, we have performed three 1500-ps PME molecular dynamics (MD) simulations, each with a different box size, on the d(CGCGA6CG)-(CGT6CGCG) DNA dodecamer. The smallest box had the DNA solvated by a layer of water molecules of at least 5 A along each orthogonal direction. The intermediate size box and the largest box had the DNA solvated by a layer of water molecules of at least 10 A and 15 A, respectively, along each orthogonal direction. The intermediate size box in the present study is similar to the box size currently chosen by most workers in the field. Based on a comparison of RMSDs and curvature for this single DNA dodecamer sequence, the larger two box sizes do not appear to afford any extra benefit over the smallest box. The implications of this finding are briefly discussed.

Base Sequence↗
PubMed1997

Effect of warmup protocol and sampling time on convergence of molecular dynamics simulations of a DNA dodecamer using AMBER 4.1 and particle-mesh Ewald method.

This report describes one 3000 ps and two 1500 ps molecular dynamic simulations on a TATA box containing dodecamer DNA duplex in a periodic box of TIP3P water molecules, using the AMBER 4.1 implementation of the particle-mesh Ewald method. We compare the effect of warmup protocol and simulation time length on the root-mean square deviation (RMSD) parameter. For the longer simulation, the RMSD computed for the 500-1000 ps time interval is representative of longer time intervals, including 500-3000 ps. The various warmup protocols do not appear to have a significant effect on the simulation results. Based on the present results, DNA sequence-dependent differences in RMSD, or related properties, should exceed two standard deviations before being attributed to non-simulation factors, such as warmup protocol and sampling time effects; we recommend a minimum criterion of at least a three standard deviation difference with a sampling period of at least 500-1000 ps. In addition, while end effects appear negligible there is a consistent dependence of RMSD on DNA helix length.

Adenoviridae↗
PubMed1997

Ab initio quantum mechanics analysis of imidazole C-H...O water hydrogen bonding and a molecular mechanics forcefield correction.

While it is well established that classical hydrogen bonds play an important role in enzyme structure, function and dynamics, the role of weaker, but 'activated' C-H donor hydrogen bonds is poorly understood. The most important such case involves histidine which often plays a direct role in enzyme catalysis and possesses the most acidic C-H donor group of the standard amino acids. In the present study, we obtained optimized geometries and hydrogen bond interaction energies for C-H...O hydrogen bonded complexes between methane, ethylene, benzene, acetylene, and imidazole with water at the MP2-FC/6-31++G(2d,2p) and MP2-FC/aug-cc-pVDZ/MP2-FC/6-31++G(2d,2p) levels of theory. A strong linear relationship is obtained between the stability of the various hydrogen bonded complexes and both separation distances for H...O and C----O. In general, these calculations indicate that C-H...O interactions can be classified as hydrogen bonding interactions, albeit significantly weaker than the classical hydrogen bonds, but significantly stronger than just van der Waals interactions. For instance, while the electronic energy of stabilization at the MP2-FC/aug-cc-pVDZ/MP2-FC/6-31++G(2d,2p) level of theory of a water O-H...O water hydrogen bond is 4.36 kcal/mol more stable than the methane C-H...O water interaction, the water-water hydrogen bond is only 2.06 kcal/mol more stable than the imidazole Ce-H...O water hydrogen bond. Neglecting this latter hydrogen bonding interaction is obviously unacceptable. We next compare the potential energy surfaces for the imidazole Ce-H...O water and imidazole Na-H...O hydrogen bonded complexes computed at the MP2/6-31++G(2d,2p) level of theory with the potential energy surface computed using the AMBER molecular mechanics program and forcefields. While the Weiner et al and Cornell et al AMBER forcefields reasonably account for the imidazole N-H...O water interaction, these forcefields do not adequately account for the imidazole Ce-H...O water hydrogen bond. A forcefield modification is offered that results in excellent agreement between the ab initio and molecular mechanics geometry and energy for this C-H...O hydrogen bonded complex.

Acetylene↗
PubMed1997

Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex between the anti HIV-1 gag ribozyme and its abortive DNA substrate manifests in the detection of a continuous track of A.T base pairs; this suggests that the interaction between the ribozyme and its DNA substrate is stronger than the one observed in the case of the free ribozyme where the bases in stem I and stem III regions interact strongly with the ribozyme core region (Sarma, R. H., et al. FEBS Letters 375, 317-23, 1995). The complex formation provides certain guidelines in the design of suitable therapeutic ribozymes. If the residues in the ribozyme stem regions interact with the conserved core, it may either prevent or interfere with the formation of a catalytically active tertiary structure.

Catalysis↗
PubMed1996

Rational approach to improving reductive catalysis by cytochrome P450cam.

Although halogenated hydrocarbons are noted for low chemical reactivity, small amounts are toxic to humans. Cytochromes P450 have been implicated in transforming these compounds to more reactive species, under anaerobic conditions, through reduction at the heme. A significant amount of effort has been directed toward turning this catalytic ability to our advantage by engineering P450 variants than can efficiently remediate these compounds in situ, before they come in contact with the human population. We have taken a 'rational' approach to this problem, in which a combination of theory and molecular modeling is applied to identify which properties of the enzyme have the greatest influence over reductive dehalogenation. Recent progress in this area is briefly reviewed. Two novel mutants, incorporating tryptophan (positions 87 and 396) and histidine (position 96, neutral and protonated) amino acid substitutions in the active site, are proposed and evaluated using molecular dynamics simulations. The upper bound on rate enhancement relative to wild-type is estimated in each mutant using electron transfer theory. The most significant rate enhancement is predicted for the His 96 mutant in the protonated state; while some His residues of certain proteins exhibit a pKa high enough to support a large protonated population, such information is not presently available for this proposed mutant.

Camphor 5-Monooxygenase↗
PubMed1996

Catalytic role of the alpha-carboxylate of the Glu residue of glutathione in glutathione S-transferases.

The recently proposed catalytic role of the alpha-carboxylate of the Glu residue of glutathione in glutathione S-transferases (Widersten et al, Biochemistry 35, 7731-7742 (1996)) was examined. Based on structural considerations, it is clear that conformational changes in both glutathione and glutathione S-transferase are required. Recent kinetic studies by Ricci and coworkers (Ricci et al, J. Biol. Chem. 271, 16187-16192 (1996) and Caccuri et al, J. Biol. Chem. 271, 16193-16198 (1996)) may provide the missing evidence for these conformational changes. Possible ways to test this hypothesis are discussed.

Carboxylic Acids↗
PubMed1996

DNA binding by TATA-box binding protein (TBP): a molecular dynamics computational study.

TATA-box binding protein (TBP) in a monomeric form and the complexes it forms with DNA have been elucidated with molecular dynamics simulations. Large TBP domain motions (bend and twist) are detected in the monomer as well as in the DNA complexes; these motions can be important for TBP binding of DNA. TBP interacts with guanine bases flanking the TATA element in the simulations of the complex; these interactions may explain the preference for guanine observed at these DNA positions. Side chains of some TBP residues at the binding interface display significant dynamic flexibility that results in 'flip-flop' contacts involving multiple base pairs of the DNA. We discuss the possible functional significance of these observations.

Arabidopsis↗
PubMed1996

A molecular dynamics study of the effect of carbon tetrachloride on enzyme structure and dynamics: subtilisin.

Developing enzymes that are functional in highly concentrated halocarbon solutions, such as carbon tetrachloride, may prove useful in the development of new strategies for environmental remediation and monitoring of pollutant plumes, as well as in developing 'green' processes. Doing so will require gaining an understanding of the underlying structural and dynamic effects on enzymes induced by such solvents. Herein we report a 714 ps molecular dynamics simulation of the enzyme subtilisin Carlsberg and its waters of crystallization in a periodic box of carbon tetrachloride. The crystal structure from aqueous solution was used as the starting structure for our simulation using the AMBER program and forcefield. The calculated time-averaged structure is similar to the aqueous X-ray structure except for significant differences in loop (or turn) regions, resulting in many extra intra-protein hydrogen bonding interactions. Since carbon tetrachloride is a non-polar solvent and cannot interact strongly with the protein and water molecules, the water molecules stay very close to the protein surface throughout the simulation. The mobility of most of the waters was therefore very low. A few water molecules underwent significant lateral motion during the simulation, but never wandered far from the protein surface. Waters were either hydrogen bonded to protein polar groups, other water and/or counterions. Some of the surface waters participated in the formation of water-mediated hydrogen bonding networks. The increase in total number of intra-protein hydrogen bonds and the formation of water-mediated hydrogen bonding networks in carbon tetrachloride is consistent with the generally observed increase in thermostability and reduced flexibility of proteins in non-aqueous solutions. Several possible carbon tetrachloride binding sites on the protein surface are predicted.

Binding Sites↗
PubMed1996

Binding free energy calculations for P450cam-substrate complexes.

A recently proposed semi-empirical method for calculating binding free energies was used to examine the binding of a variety of substrates to cytochrome P450cam. For a set of 11 different potential substrates of cytochrome P450cam, both the absolute and relative binding free energies were generally well reproduced. The mean error in the calculated absolute binding free energy for all 11 compounds is 0.55 kcal/mol. Forty-eight out of 55 calculated relative binding free energies have the correct sign and the mean unsigned error between calculated and experimental relative binding free energies is 0.77 kcal/mol. For one substrate, thiocamphor, the effect of substrate orientation on the calculated binding free energy was examined. The ability of this method to predict the effect of active site mutations was also examined in two cases.

Camphor↗
PubMed1995

Stereoselective hydroxylation of norcamphor by cytochrome P450cam. Experimental verification of molecular dynamics simulations.

The stereoselectivity of cytochrome P450cam hydroxylation has been investigated with the enantiomerically pure substrate analog norcamphor. (1R)- and (1S)-norcamphor (> 92 enantiomeric excess) were characterized in the hydroxylation reaction with cytochrome P450cam with respect to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation. The experimental results demonstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M. B., and Ornstein, R. L. (1993) J. Comput. Chem. 14, 541-548); these simulation results are now compared with simulations involving a ferryl oxygen intermediate. Hydroxylation of (1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the same products. With the exception of the regiospecificity, all of the reaction and physical parameters are similar for each enantiomer of norcamphor. These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining the average orientation of this substrate in the active site and suggests that hydrogen bonding interactions can aid in directing the regiospecificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450cam.

Binding Sites↗
PubMed1995

Secondary structure in solution of two anti-HIV-1 hammerhead ribozymes as investigated by two-dimensional 1H 500 MHz NMR spectroscopy in water.

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

Base Sequence↗