Papers by Donald Bashford
Frontiers in bioscience : a journal and virtual library, 2004
The use of macroscopic electrostatic models to calculate the relative energetics of protonation s... more The use of macroscopic electrostatic models to calculate the relative energetics of protonation states and the pH-titration properties of ionizable groups in proteins is described. These methods treat the protein as an irregularly-shaped low-dielectric object containing embedded atomic charges immersed in a high-dielectric (solvent) medium. The energetics of altering protonation states then involves the electrostatic work of altering the embedded atomic charges. The governing electrostatic equation is either the Poisson or linearized Poisson-Boltzmann equation, which generally requires numerical solution. A tutorial approach is taken, the main aim of which is a thorough understanding of the method.
Use of Broken-Symmetry Density Functional Theory To Characterize the IspH Oxidized State: Implications for IspH Mechanism and Inhibition
Journal of chemical theory and computation, Jan 9, 2014
With current therapies becoming less efficacious due to increased drug resistance, new inhibitors... more With current therapies becoming less efficacious due to increased drug resistance, new inhibitors of both bacterial and malarial targets are desperately needed. The recently discovered methylerythritol phosphate (MEP) pathway for isoprenoid synthesis provides novel targets for the development of such drugs. Particular attention has focused on the IspH protein, the final enzyme in the MEP pathway, which uses its [4Fe-4S] cluster to catalyze the formation of the isoprenoid precursors IPP and DMAPP from HMBPP. IspH catalysis is achieved via a 2e (-)/2H(+) reductive dehydroxylation of HMBPP; the mechanism by which catalysis is achieved, however, is highly controversial. The work presented herein provides the first step in assessing different routes to catalysis by using computational methods. By performing broken-symmetry density functional theory (BS-DFT) calculations that employ both the conductor-like screening solvation model (DFT/COSMO) and a finite-difference Poisson-Boltzmann self-consistent reaction field methodology (DFT/SCRF), we evaluate geometries, energies, and Mössbauer signatures of the different protonation states that may exist in the oxidized state of the IspH catalytic cycle. From DFT/SCRF computations performed on the oxidized state, we find a state where the substrate, HMBPP, coordinates the apical iron in the [4Fe-4S] cluster as an alcohol group (ROH) to be one of two, isoenergetic, lowest-energy states. In this state, the HMBPP pyrophosphate moiety and an adjacent glutamate residue (E126) are both fully deprotonated, making the active site highly anionic. Our findings that this low-energy state also matches the experimental geometry of the active site and that its computed isomer shifts agree with experiment validate the use of the DFT/SCRF method to assess relative energies along the IspH reaction pathway. Additional studies of IspH catalytic intermediates are currently being pursued.
Implicit solvation models provide, for many applications, a reasonably accurate and computational... more Implicit solvation models provide, for many applications, a reasonably accurate and computationally effective way to describe the electrostatics of aqueous solvation. Here, a popular analytical Generalized Born (GB) solvation model is modified to improve its accuracy in calculating the solvent polarization part of free energy changes in large-scale conformational transitions, such as protein folding. In contrast to an earlier GB model (implemented in the AMBER-6 program), the improved version does not overstabilize the native structures relative to the finite-difference Poisson-Boltzmann continuum treatment. In addition to improving the energy balance between folded and unfolded conformers, the algorithm (available in the AMBER-7 and NAB molecular modeling packages) is shown to perform well in more than 50 ns of native-state molecular dynamics (MD) simulations of thioredoxin, protein-A, and ubiquitin, as well as in a simulation of Barnase/Barstar complex formation. For thioredoxin, various combinations of input parameters have been explored, such as the underlying gas-phase force fields and the atomic radii. The best performance is achieved with a previously proposed modification to the torsional potential in the Amber ff99 force field, which yields stable native trajectories for all of the tested proteins, with backbone root-mean-square deviations from the native structures being ϳ1.5 Å after 6 ns of simulation time. The structure of Barnase/Barstar complex is regenerated, starting from an unbound state, to within 1.9 Å relative to the crystal structure of the complex.
Quantitative structure–activity relationship (QSAR) for a series of novel cannabinoid derivatives using descriptors derived from semi-empirical quantum-chemical calculations
Density Functional and Electrostatic Calculations of Manganese Superoxide Dismutase Active Site Complexes in Protein Environments
Inorganic Chemistry, 1999
Density functional and electrostatic methods have been applied to calculate active site geometrie... more Density functional and electrostatic methods have been applied to calculate active site geometries and the redox potential of manganese superoxide dismutase (MnSOD). The initial active site clusters were built up by including only first-shell side chain ligands and then augmented by second-shell ligands. The density functional optimized Mn-ligand bond lengths for the reduced complexes in general compared fairly well with protein crystallography data; however, large deviations for calculated Mn-OH distances were found for the oxidized active site clusters. Our calculations suggest that this deviation can be attributed to the redox heterogeneity of the oxidized protein in X-ray crystallography studies. The redox potential was calculated by treating the protein environment and the solvent bulk by a semimacroscopic electrostatic model. The protein structures were taken from the Thermus thermophilus enzyme. The calculated coupled redox potentials converge toward experimental values with increasing size of the active site cluster models, and the final calculated value was +0.06 V, compared to experimental values of +0.26 V determined for Bacillus stearothermophilus and +0.31 V in Escherichia coli enzymes. Using an energy decomposition scheme, the effects of the second-shell ligands and the protein and reaction fields have been analyzed.
Inorganic Chemistry, 1996
Density functional and continuum dielectric theories have been combined to calculate molecular pr... more Density functional and continuum dielectric theories have been combined to calculate molecular properties such as hydration enthalpies, redox potentials, and absolute pK a values of transition metal cations in solution. The discrete cluster model, which is treated explicitly by density functional theory, includes six waters in the first hydration shell and another twelve waters in the second shell. The solvent reaction field is obtained from a finite-difference solution to the Poisson-Boltzmann equation and is coupled to the nonlocal density functional calculation in a self-consistent way. The calculated hydration enthalpies are 409, 1073, 431, and 1046 kcal/mol for Mn 2+ , Mn 3+ , Fe 2+ , and Fe 3+ , respectively, comparing fairly well to the experimental measurements of 440, 1087, 465, and 1060 kcal/mol. The calculated redox potentials for the Mn 2+ /Mn 3+ and Fe 2+ /Fe 3+ pairs are 1.59 and 1.06 V, respectively, in good agreement with the experimental values of 1.56 and 0.77 V. The computed absolute pK a values, 14.0, -6.5, 9.0, and -4.0 for Mn 2+ , Mn 3+ , Fe 2+ , and Fe 3+ , respectively, deviate significantly from the experimental results of 10.6, 0.1, 9.5, and 2.2 but show the proper behavior with changes in oxidation state and metal type. The calculated redox potentials and pK a values appear to converge toward the experimental data with increasing size of the cluster models. For such highly charged cations, the second hydration shell in the cluster model is indispensable, since this buffer shell retains strong hydrogen bonds and electron transfer between the inner and outer shells as well as the solute-solvent dispersion interaction. X
On the Role of the Conserved Aspartate in the Hydrolysis of the Phosphocysteine Intermediate of the Low Molecular Weight Tyrosine Phosphatase
Journal of The American Chemical Society - J AM CHEM SOC, 2004
The usual rate-determining step in the catalytic mechanism of the low molecular weight tyrosine p... more The usual rate-determining step in the catalytic mechanism of the low molecular weight tyrosine phosphatases involves the hydrolysis of a phosphocysteine intermediate. To explain this hydrolysis, general base-catalyzed attack of water by the anion of a conserved aspartic acid has sometimes been invoked. However, experimental measurements of solvent deuterium kinetic isotope effects for this enzyme do not reveal a rate-limiting proton transfer accompanying dephosphorylation. Moreover, base activation of water is difficult to reconcile with the known gas-phase proton affinities and solution phase pK(a)'s of aspartic acid and water. Alternatively, hydrolysis could proceed by a direct nucleophilic attack by a water molecule. To understand the hydrolysis mechanism, we have used high-level density functional methods of quantum chemistry combined with continuum electrostatics models of the protein and the solvent. Our calculations do not support a catalytic activation of water by the aspartate. Instead, they indicate that the water oxygen directly attacks the phosphorus, with the aspartate residue acting as a H-bond acceptor. In the transition state, the water protons are still bound to the oxygen. Beyond the transition state, the barrier to proton transfer to the base is greatly diminished; the aspartate can abstract a proton only after the transition state, a result consistent with experimental solvent isotope effects for this enzyme and with established precedents for phosphomonoester hydrolysis.
Proteins-structure Function and Bioinformatics, 2001
In calculations involving many displacements of an interacting pair of biomolecules, such as brow... more In calculations involving many displacements of an interacting pair of biomolecules, such as brownian dynamics, the docking of a substrate/ligand to an enzyme/receptor, or the screening of a large number of ligands as prospective inhibitors for a particular receptor site, there is a need for rapid evaluation of the desolvation penalties of the interacting pair. Although continuum electrostatic treatments with distinct dielectric constants for solute and solvent provide an account of the electrostatics of solvation and desolvation, it is necessary to re-solve the Poisson equation, at considerable computational cost, for each displacement of the interacting pair. We present a new method that uses a formulation of continuum electrostatic solvation in terms of the solvation energy density and approximates desolvation in terms of the occlusion of this density. We call it the SEDO approximation. It avoids the need to re-solve the Poisson equation, as desolvation is now estimated by an integral over the occluded volume. Test calculations are presented for some simple model systems and for some real systems that have previously been studied using the Poisson equation approach: MHC class I protein-peptide complexes and a congeneric series of human immunodeficiency virus type 1 (HIV-1) protease-ligand complexes. For most of the systems considered, the trends and magnitudes of the desolvation component of interaction energies obtained using the SEDO approximation are in reasonable correlation with those obtained by re-solving the Poisson equation. In most cases, the error introduced by the SEDO approximation is much less than that of the often-used test-charge approximation for the charge-charge components of intermolecular interactions. Proteins 2001;43:12-27.
Journal of The American Chemical Society, 1997
The tetrapeptides APGD and APGN are known by NMR analysis to adopt reverse turn conformations to ... more The tetrapeptides APGD and APGN are known by NMR analysis to adopt reverse turn conformations to a significant degree in aqueous solution. We have carried out a 7.7 ns molecular dynamics simulation of Ace-APGD-NHMe in explicit water, and have analyzed the energetics of snapshots from this simulation in terms of a molecular mechanics energy function, estimates of solvation free energy based on numerical solutions of the Poisson-Boltzmann equation (in which the solvent is treated as a high-dielectric continuum), and an estimate of chain entropy effects derived from a systematic search procedure. In the unconstrained trajectory, 17 transitions occur between turn and extended conformers, suggesting that the free energy profile is nearly flat and that the simulation is moderatelywell-equilibrated with respect to this transition; the turn population found is within the experimental range. The potential of mean force, constructed as the sum of solute force-field energies, continuum solvation, and hard-sphere chain entropy, agrees with that computed directly from the simulation to within 2 kcal/mol across the entire range of configurations sampled. The study has been extended to the tetrapeptide APGN by repeating the energetic analysis with an Asn side chain replacing the Asp in the APGD snapshots. A comparison of energetics with Asp and Asn side chains shows a complex interplay among vacuum electrostatic terms, dielectric screening terms, and solvation free energy terms such that the net effect of side chain substitution on turn formation is very small. Prospects for application of this sort of analysis to other peptide and protein conformational problems are discussed.
Stabilization of charges and protonation states in the active site of the protein tyrosine phosphatases: A computational study
The Journal of Physical …, 2000
The initial step of the dephosphorylation of phosphotyrosine by the protein tyrosine phosphatases... more The initial step of the dephosphorylation of phosphotyrosine by the protein tyrosine phosphatases (PTPases) requires an active-site cysteine residue to be in its deprotonated (thiolate) form so as to make a nucleophilic attack on the phosphate to form a ...
Incorporating solvation effects into density functional electronic structure calculations
The Journal of Physical …, 1994
Page 1. J. Phys. Chem. 1994, 98, 11059-11068 11059 Incorporating Solvation Effects into Density F... more Page 1. J. Phys. Chem. 1994, 98, 11059-11068 11059 Incorporating Solvation Effects into Density Functional Electronic Structure Calculations Jun L. Chen, Louis Noodleman; David A. Case,* and Donald Bashford* Department ...
Generalized Born (GB) models provide, for many applications, an accurate and computationally faci... more Generalized Born (GB) models provide, for many applications, an accurate and computationally facile estimate of the electrostatic contribution to aqueous solvation. The GB models involve two main types of approximations relative to the Poisson equation (PE) theory on which they are based. First, the self-energy contributions of individual atoms are estimated and expressed as "effective Born radii." Next, the atom-pair contributions are estimated by an analytical function f GB that depends upon the effective Born radii and interatomic distance of the atom pairs. Here, the relative impacts of these approximations are investigated by calculating "perfect" effective Born radii from PE theory, and enquiring as to how well the atom-pairwise energy terms from a GB model using these perfect radii in the standard f GB function duplicate the equivalent terms from PE theory. In tests on several biological macromolecules, the use of these perfect radii greatly increases the accuracy of the atom-pair terms; that is, the standard form of f GB performs quite well. The remaining small error has a systematic and a random component. The latter cannot be removed without significantly increasing the complexity of the GB model, but an alternative choice of f GB can reduce the systematic part. A molecular dynamics simulation using a perfect-radii GB model compares favorably with simulations using conventional GB, even though the radii remain fixed in the former. These results quantify, for the GB field, the importance of getting the effective Born radii right; indeed, with perfect radii, the GB model gives a very good approximation to the underlying PE theory for a variety of biomacromolecular types and conformations.
The analytic generalized Born approximation is an efficient electrostatic model that describes mo... more The analytic generalized Born approximation is an efficient electrostatic model that describes molecules in solution. Here it is modified to permit a more accurate description of large macromolecules, while its established performance on small compounds is nearly unaffected. The modified model is also adapted to describe molecules with an interior dielectric constant not equal to unity. The model is tested by computations of pK shifts for a number of titratable residues in lysozyme, myoglobin, and bacteriorhodopsin. In general, except for some deeply buried residues of bacteriorhodopsin, the results show reasonable agreement with both experimental data and calculations based on numerical solution of the Poisson-Boltzmann equation. A very close agreement between the two models is obtained in an application to the prediction of the pK shifts associated with conformational change. The calculations based on this version of the generalized Born approximation are much faster than finite difference solutions of the Poisson-Boltzmann equation, which makes the present method useful for a variety of other applications where computational time is a critical factor. The model may also be integrated into molecular dynamics programs to replace explicit solvent simulations which are particularly time-consuming for large molecules.
Journal of the American Chemical Society, Jul 9, 2003
The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioes... more The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioester chromophore that absorbs blue light, initiating a photocycle involving a series of conformational changes. Here, we present computational studies to resolve uncertainties and controversies concerning the correspondence between atomic structures and spectroscopic measurements on early photocycle intermediates. The initial nanoseconds of the PYP photocycle are examined using time-dependent density functional theory (TDDFT) to calculate the energy profiles for chromophore photoisomerization and proton transfer, and to calculate excitation energies to identify photocycle intermediates. The calculated potential energy surface for photoisomerization matches key, experimentally determined, spectral parameters. The calculated excitation energy of the photocycle intermediate cryogenically trapped in a crystal structure by Genick et al. [Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D. Nature 1998, 392, 206-209] supports its assignment to the PYPB (I0) intermediate. Differences between the time-resolved room temperature (298 K) spectrum of the PYPB intermediate and its low temperature (77 K) absorbance are attributed to a predominantly deprotonated chromophore in the former and protonated chromophore in the latter. This contrasts with the widely held belief that chromophore protonation does not occur until after the PYP L (I1 or pR) intermediate. The structure of the chromophore in the PYPL intermediate is determined computationally and shown to be deprotonated, in agreement with experiment. Calculations based on our PYP B and PYPL models lead to insights concerning the PYPBL intermediate, observed only at low temperature.
Identification and Characterization of an Allosteric Inhibitory Site on Dihydropteroate Synthase
ACS Chemical Biology, 2014
The declining effectiveness of current antibiotics due to the emergence of resistant bacterial st... more The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development. Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and quasi-harmonic analyses suggest that the effect of inhibitor binding is transmitted from the dimer interface to the active-site loops that are known to assume an obligatory ordered substructure during catalysis. Together with the kinetics results, these structural and dynamics data suggest an inhibitory mechanism in which binding at the dimer interface impacts loop movements that are required for product release. Our results potentially provide a novel target site for the development of new antibiotics.
Blood, Jan 21, 2014
HLA-DRB1*07:01 is associated with asparaginase hypersensitivity and antiasparaginase antibodies.
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Papers by Donald Bashford