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J W Brady

Publications and source records attributed to J W Brady.

At least 19 recordsLinked to original sources
PubMed2006

Nanometer-scale ion aggregates in aqueous electrolyte solutions: guanidinium carbonate.

Neutron diffraction with isotopic substitution (NDIS) experiments and molecular dynamics (MD) simulations have been used to characterize the structure of aqueous guanidinium carbonate (Gdm2CO3) solutions. The MD simulations found very strong hetero-ion pairing in Gdm2CO3 solution and were used to determine the best structural experiment to demonstrate this ion pairing. The NDIS experiments confirm the most significant feature of the MD simulation, which is the existence of strong hetero-ion pairing between the Gdm+ and CO3(2-) ions. The neutron structural data also support the most interesting feature of the MD simulation, that the hetero-ion pairing is sufficiently strong as to lead to nanometer-scale aggregation of the ions. The presence of such clustering on the nanometer length scale was then confirmed using small-angle neutron scattering experiments. Taken together, the experiment and simulation suggest a molecular-level explanation for the contrasting denaturant properties of guanidinium salts in solution.

Electrolytes↗
PubMed2005

Nanometer-scale ion aggregates in aqueous electrolyte solutions: guanidinium sulfate and guanidinium thiocyanate.

Neutron diffraction experiments and molecular dynamics simulations are used to study the structure of aqueous solutions of two electrolytes: guanidinium sulfate (a mild protein conformation stabilizer) and guanidinium thiocyanate (a powerful denaturant). The MD simulations find the unexpected result that in the Gdm2SO4 solution the ions aggregated into mesoscopic (nanometer-scale) clusters, while no such aggregation is found in the GdmSCN solution. The neutron diffraction studies, the most direct experimental probe of solution structure, provide corroborating evidence that the predicted very strong ion pairing does occur in solutions of 1.5 m Gdm2SO4 but not in 3 m solutions of GdmSCN. A mechanism is proposed as to how this mesoscopic solution structure affects solution denaturant properties and suggests an explanation for the Hofmeister ordering of these solutions in terms of this ion pairing and the ability of sulfate to reverse the denaturant power of guanidinium.

Electrolytes↗
PubMed2005

Structure of aqueous glucose solutions as determined by neutron diffraction with isotopic substitution experiments and molecular dynamics calculations.

Neutron diffraction with isotopic substitution (NDIS) experiments and molecular dynamics (MD) simulations have been used to examine the structuring of solvent around d-glucose in aqueous solution. As expected, no significant tendency for glucose molecules to aggregate was found in either the experiments or the simulation. To the extent that solute pairing does occur as the result of the high concentration, it was found to take place through hydroxyl-hydroxyl hydrogen bonds, in competition with water molecules for the same hydrogen-bonding sites. A detailed analysis of the hydrogen-bonding patterns occurring in the simulations found that the sugar hydroxyl groups are more efficient hydrogen bond donors than acceptors. From the comparison of the MD and NDIS data, it was found that while the modeling generally does a satisfactory job in reproducing the experimental data the force fields may produce sugar rings that are too rigid and thus may require future revisions.

Computer Simulation↗
PubMed2003

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2).

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.

Actinomycetales↗
PubMed2003

Energetics for displacing a single chain from the surface of microcrystalline cellulose into the active site of Acidothermus cellulolyticus Cel5A.

A series of molecular mechanics calculations were used to analyze the energetics for moving a single polysaccharide chain from the surface of microcrystalline cellulose into the binding cleft of the Cel5A cellulase from Acidothermus cellulolyticus. A build-up procedure was used to model the placement of a 12-residue oligosaccharide chain along the surface of the enzyme, using as a guide the four residues of the tetrasaccharide substrate co-crystallized with the protein in the crystallographic structure determination. The position of this 12-residue oligosaccharide was used to orient the enzyme properly above two different surfaces of cellulose 1beta, the (1,0,0) and the (1,1,0) faces of the crystal. Constrained molecular dynamics simulations were then used to pull a target chain directly below the enzyme up out of the crystal surface and into the binding groove. The energetics for this process were favorable for both faces, with the step face being more favorable than the planar face, implying that this surface could be hydrolyzed more readily.

Actinobacteria↗
PubMed2002

Carbohydrate solution simulations: producing a force field with experimentally consistent primary alcohol rotational frequencies and populations.

We present a CHARMM Carbohydrate Solution Force Field (CSFF) suitable for nanosecond molecular dynamics computer simulations. The force field was derived from a recently published sugar parameter set.1 Dihedral angle parameters for the primary alcohol as well as the secondary hydroxyl groups were adjusted. Free energy profiles of the hydroxymethyl group for two monosaccharides (beta-D-glucose and beta-D-galactose) were calculated using the new parameter set and compared with similar force fields. Equilibrium rotamer populations obtained from the CSFF are in excellent agreement with NMR data (glucose gg:gt:tg approximately 66:33:1 and galactose gg:gt:tg approximately 4:75:21). In addition, the primary alcohol rotational frequency is on the nanosecond time scale, which conforms to experimental observations. Equilibrium population distributions of the primary alcohol conformers for glucose and galactose are reached within 10 nanoseconds of molecular dynamics simulations. In addition, gas phase vibrational frequencies computed for beta-D-glucose using this force field compare well with experimental frequencies. Carbohydrate parameter sets that produce both conformational energies and rotational frequencies for the pyranose primary alcohol group that are consistent with experimental observations should allow for increased accuracy in modeling the flexibility of biologically important (1-6)-linked saccharides in solution.

Alcohols↗
PubMed1997

Molecular dynamics simulations of the N-linked oligosaccharide of the lectin from Erythrina corallodendron.

Molecular dynamics simulations have been used to model the flexibility of the seven-sugar oligosaccharide of the lectin from Erythrina corallodendron in three separate simulations: one of the isolated oligosaccharide in vacuo, one of the oligosaccharide in solution and one of the oligosaccharide linked to the protein in solution. Adiabatic conformational energy maps were prepared for each of the disaccharide linkages as a means of interpreting the observed dynamics and conformational averages in terms of intramolecular energy. The inclusion of aqueous solvent molecules appears to be necessary to reproduce the experimental conformational behavior, which also cannot be predicted well from conformational energy maps for the disaccharide linkages alone. The crystallographically determined conformation does not appear to be induced by the crystal dimerization, but is rather stable in solution. The build-up of fluctuations along the successive linkages of the oligosaccharide is significant and would be sufficient to prevent branch residues from being located in most crystal structure determinations. Good general agreement between the calculated solution structure and the average structure determined by NMR was found for most of the oligosaccharide linkages.

Carbohydrate Conformation↗
PubMed1996

The effect of hydration upon the conformation and dynamics of neocarrabiose, a repeat unit of beta-carrageenan.

Molecular mechanics calculations have been performed for the disaccharide neocarrabiose, one of the repeat units of beta-carrageenan, as a general model for the (1-->3)-linkage in the carrageenans. An adiabatic conformational energy map for this molecule has been prepared by constrained energy minimization and compared to previously reported relaxed maps. Neither the experimentally determined crystal structure of neocarrabiose nor the fiber diffraction conformation of iota-carrageenan is a low energy conformation on the relaxed Ramachandran map. Molecular dynamics simulations in vacuum produced trajectories consistent with this relaxed vacuum surface. However, a simulation with explicitly included solvent water molecules produced a trajectory that remained in the region of the two experimental structures. This dramatic solvation effect is apparently the result of the breaking of an interring hydrogen bond between the O2 hydroxyl groups of neocarrabiose as both groups hydrogen bond to solvent.

Carbohydrate Conformation↗
PubMed1995

Conformational modeling of substrate binding to endocellulase E2 from Thermomonospora fusca.

Molecular mechanics calculations have been used to place a cellotetraose substrate into the active site of the crystallographically determined structure of endocellulase E2 from Thermomonospora fusca. In the lowest energy model structure, the second residue of the substrate oligosaccharide is tilted away from the planar ribbon geometry of cellulose as it is in the X-ray structure of the E2cd-cellobiose co-crystal. This tilt is the result of the topology of the binding site, and results in several strong carbohydrate-protein hydrogen bonds. The tilting produces a twisting of the glycosidic linkage of the cleavage site between residues two and three. In the predicted enzyme-substrate complex both of the Asp residues believed to function in general acid and base roles in the previously proposed model for the mechanism are distant from the bond being cleaved. Molecular dynamics simulations of the complex were conducted, and while the putative catalytic Asp residues remained distant from the cleavage site, the proton of Tyr73 briefly came within van der Waals contact of the linkage oxygen.

Actinomycetales↗
PubMed1994

Computer simulations of cyclic and acyclic cholinergic agonists: conformational search and molecular dynamics simulations.

Molecular dynamics simulations have been performed on aqueous solutions of two chemically similar nicotinic cholinergic agonists in order to compare their structural and dynamical differences. The cyclic 1,1-dimethyl-4-acetylpiperazinium iodide (HPIP) molecule was previously shown to be a strong agonist for nicotinic acetylcholine receptors (McGroddy et al., 1993), while the acyclic N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide (HTED) derivative is much less potent. These differences were expected to arise from differences in the solution structures and internal dynamics of the two molecules. HPIP was originally thought to be relatively rigid; however, molecular dynamics simulations suggest that the acetyl portion of the molecule undergoes significant ring dynamics on a psec timescale. The less constrained HTED molecule is relatively rigid, with only one transition observed about any of the major dihedrals in four 100 psec simulations, each started from a different conformation. The average structures obtained from the simulations are very similar to the starting minimized structure in each case, except for the HTED simulation where a single rotation about the N-C-C-N(+) backbone occurred. In each case, HTED had three to five more water molecules in its primary solvation shell than HPIP, indicating that differences in the energetics of desolvation before binding may partially explain the increased potency of HPIP as compared to HTED.

Biophysical Phenomena↗
PubMed1994

Thermostable variants of bovine beta-lactoglobulin.

The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond. Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121. We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C. Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein. These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method. Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping. In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized. The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.

Amino Acid Sequence↗
PubMed1993

Molecular dynamics simulations of a winter flounder "antifreeze" polypeptide in aqueous solution.

A winter flounder antifreeze polypeptide (HPLC-6) has been studied in vacuo and in aqueous solution using molecular dynamics computer simulation techniques. The helical conformation of this polypeptide was found to be stable both in vacuum and in solution. The major stabilizing interactions were found to be the main-chain hydrogen bonds, a salt-bridge interaction, and solute-solvent hydrogen bonds. A significant bending in the middle of the polypeptide chain was observed both in vacuo and in solvent at 300 K. Possible causes of the bending are discussed. From simulations of mutant polypeptide molecules in vacuo, it is concluded that the bend in the native polypeptide was caused by side chain to backbone hydrogen bond competition involving the Thr 24 side chain and facilitated by strains on the helix resulting from the Lys 18-Glu 22 salt bridge.

Amino Acid Sequence↗
PubMed1992

Molecular dynamics simulations of the whey protein beta-lactoglobulin.

Molecular dynamics simulations have been used to model the motions and conformational behavior of the whey protein bovine beta-lactoglobulin. Simulations were performed for the protein by itself and complexed to a single retinol ligand located in a putative interior binding pocket. In the absence of the retinol ligand, the backbone loops around the opening of this interior pocket shifted inward to partially close off this cavity, similar to the shifts observed in previously reported molecular dynamics simulations of the uncomplexed form of the homologous retinol binding protein. The protein complexed with retinol does not exhibit the same conformational shifts. Conformational changes of this type could serve as a recognition signal allowing in vivo discrimination between the free and retinol complexed forms of the beta-lactoglobulin molecule. The unusual bending of the single alpha-helix observed in the simulations of retinol binding protein were not observed in the present calculations.

Animals↗
PubMed1991

Molecular dynamics simulation of the aqueous solvation of sugars.

Recently, several molecular dynamics simulations of the aqueous solvation of carbohydrates have been reported. These studies represent the first theoretical picture available of the microscopic character of sugar solutions, and may provide explanations of the unusual and complex behavior of this class of molecules in solution. This paper will discuss two MD simulations of D-glucopyranose, including a free energy perturbation calculation of the anomeric free energy difference. Solvation was found to have little effect upon the mean conformational structure of the pyranoid rings, but the presence of solvent significantly affected the motions and orientations of the exocyclic groups. Adjacent functional groups of the sugar rings were found to mutually perturb one another's hydration, depending upon the local stereochemistry, which may prove to play a part in the observed anomeric preferences of the sugars. From a component analysis of the free energy of solvation of the two anomers of D-glucopyranose, it was found that a large solvation term favors the beta anomer, which is the form found to be preferred in aqueous solution.

Carbohydrates↗
PubMed1988

A revised potential-energy surface for molecular mechanics studies of carbohydrates.

A revised CHARMM-type molecular mechanics potential-energy function has been developed for use in the dynamical simulation of simple carbohydrates in aqueous solution. Atomic charges used in this parameterization were taken to be those previously determined to be appropriate for hydrogen-bonded systems, and the various force-constants were selected by the nonlinear least-squares matching of the calculated normal-mode frequencies and minimum-energy structure to experiment as a function of the parameter set. The new function was found to represent the vibrational spectrum and ring pucker of alpha-D-glucopyranose as well as previously studied potentials, while incorporating the charges necessary for the simulation of condensed phases. Molecular dynamics simulations of the motions of alpha-D-glucopyranose in vacuo in both the 1C4 and 4C1 conformation were conducted, and compared to the results of previous simulations using another potential-energy function. The revised potential function was found to produce a D-glucose molecule less flexible in vacuo than had been previously observed.

Carbohydrates↗