Brian Dominy | Clemson University (original) (raw)

Papers by Brian Dominy

Communications in Computational Physics

Salt influences protein stability through electrostatic mechanisms as well as through nonpolar Ho... more Salt influences protein stability through electrostatic mechanisms as well as through nonpolar Hofmeister effects. In the present work, a continuum solvation based model is developed to explore the impact of salt on protein stability. This model relies on a traditional Poisson-Boltzmann (PB) term to describe the polar or electrostatic effects of salt, and a surface area dependent term containing a salt concentration dependent microscopic surface tension function to capture the non-polar Hofmeister effects. The model is first validated against a series of cold-shock protein variants whose salt-dependent protein fold stability profiles have been previously determined experimentally. The approach is then applied to HIV-1 protease in order to explain an experimentally observed enhancement in stability and activity at high (1M) NaCl concentration. The inclusion of the salt-dependent non-polar term brings the model into quantitative agreement with experiment, and provides the basis for fu...

Communications in Computational Physics

Recently, it was demonstrated that implicit solvent models were capable of generating stable B-fo... more Recently, it was demonstrated that implicit solvent models were capable of generating stable B-form DNA structures. Specifically, generalized Born (GB) implicit solvent models have improved regarding the solvation of conformational sampling of DNA [1,2]. Here, we examine the performance of the GBSW and GBMV models in CHARMM for characterizing base flipping free energy profiles of undamaged and damaged DNA bases. Umbrella sampling of the base flipping process was performed for the bases cytosine, uracil and xanthine. The umbrella sampling simulations were carried-out with both explicit (TIP3P) and implicit (GB) solvent in order to establish the impact of the solvent model on base flipping. Overall, base flipping potential of mean force (PMF) profiles generated with GB solvent resulted in a greater free energy difference of flipping than profiles generated with TIP3P. One of the significant differences between implicit and explicit solvent models is the approximation of solute-solvent...

Journal of Computational Chemistry

One of the anthrax receptors, tumor endothelial marker 8 (TEM8), is reported to be a potential an... more One of the anthrax receptors, tumor endothelial marker 8 (TEM8), is reported to be a potential anticancer target due to its over‐expression during tumor angiogenesis. To extend our BioLayer Interferometry study in PA‐TEM8 binding, we present a computational approach to reveal the role of an integral metal ion on receptor structure and binding thermodynamics. We estimated the interaction energy between PA and TEM8 using computer simulation. Consistent with experimental study, computational results indicate the metal ion in TEM8 contributes significantly to the binding affinity, and PA‐TEM8 binding is more favorable in the presence of Mg2+ than Ca2+. Further, computational analysis suggests that the differences in PA‐TEM8 binding affinity are comparable to the closely related integrin proteins. The conformation change, which linked to changes in activity of integrins, was not found in TEM8. In the present of Mg2+, TEM8 remains in a conformation analogous to an integrin open (high‐affinity) conformation. © 2017 Wiley Periodicals, Inc.

Scientific reports, Apr 11, 2017

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Fami... more Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in m...

Communications in Computational Physics

Recently, it was demonstrated that implicit solvent models were capable of generating stable B-fo... more Recently, it was demonstrated that implicit solvent models were capable of generating stable B-form DNA structures. Specifically, generalized Born (GB) implicit solvent models have improved regarding the solvation of conformational sampling of DNA [1, 2]. Here, we examine the performance of the GBSW and GBMV models in CHARMM for characterizing base flipping free energy profiles of undamaged and damaged DNA bases. Umbrella sampling of the base flipping process was performed for the bases cytosine, uracil and xanthine. The umbrella sampling simulations were carried-out with both explicit (TIP3P) and implicit (GB) solvent in order to establish the impact of the solvent model on base flipping. Overall, base flipping potential of mean force (PMF) profiles generated with GB solvent resulted in a greater free energy differ-ence of flipping than profiles generated with TIP3P. One of the significant differences between implicit and explicit solvent models is the approximation of solute-solve...

Nucleic acids research, 2015

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-s... more The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb...

The Journal of biological chemistry, Jan 9, 2011

DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigation... more DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigations identified five families within the uracil-DNA glycosylase (UDG) superfamily. All enzymes within the superfamily studied thus far exhibit uracil-DNA glycosylase activity. Here we identify a new class of DNA glycosylases in the UDG superfamily that lacks UDG activity. Instead, these enzymes act as hypoxanthine-DNA glycosylases in vitro and in vivo. Molecular modeling and structure-guided mutational analysis allowed us to identify a unique catalytic center in this class of DNA glycosylases. Based on unprecedented biochemical properties and phylogenetic analysis, we propose this new class of DNA repair glycosylases that exists in bacteria, archaea, and eukaryotes as family 6 and designate it as the hypoxanthine-DNA glycosylase family. This study demonstrates the structural evolvability that underlies substrate specificity and catalytic flexibility in the evolution of enzymatic function.

The Journal of biological chemistry, Jan 27, 2014

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that fa... more UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb re...

The Journal of Physical Chemistry B, 1999

... This factor remains limiting and to date has hampered the quest for relevant studies of prote... more ... This factor remains limiting and to date has hampered the quest for relevant studies of protein folding and dynamics on microsecond time ... 7. One model that provides an approximate continuum solvent representation is the generalized Born (GB) model introduced by Still and co ...

Proteins: Structure, Function, and Genetics, 1999

A protocol for the rapid energetic analysis of protein-ligand complexes has been developed. This ... more A protocol for the rapid energetic analysis of protein-ligand complexes has been developed. This protocol involves the generation of protein-ligand complex ensembles followed by an analysis of the binding free energy components. We apply this methodology toward understanding the origin of binding specificity within the human immunodeficiency virus/feline immunodeficiency virus (HIV/FIV) protease system, a model system for drug resistance studies. A distinct difference in the internal strain of an inhibitor within each protein environment clearly favors the HIV protease complex, as observed experimentally. Our analysis also predicts that residues within the S2-S3 pockets of the FIV protease active site are responsible for this strain. Close examination of the active site residue contributions to interaction energy and desolvation energy identifies specific amino acids that may also play a role in determining the binding preferences of these two enzymes. Proteins 1999;36:318-331.

Proteins: Structure, Function, and Genetics, 1999

The relationship between the unfolding pseudo free energies of reduced and detailed atomic models... more The relationship between the unfolding pseudo free energies of reduced and detailed atomic models of the GCN4 leucine zipper is examined. Starting from the native crystal structure, a large number of conformations ranging from folded to unfolded were generated by all-atom molecular dynamics unfolding simulations in an aqueous environment at elevated temperatures. For the detailed atomic model, the pseudo free energies are obtained by combining the CHARMM all-atom potential with a solvation component from the generalized Born, surface accessibility, GB/SA, model. Reduced model energies were evaluated using a knowledge-based potential. Both energies are highly correlated. In addition, both show a good correlation with the root mean square deviation, RMSD, of the backbone from native. These results suggest that knowledge-based potentials are capable of describing at least some of the properties of the folded as well as the unfolded states of proteins, even though they are derived from a database of native protein structures. Since only conformations generated from an unfolding simulation are used, we cannot assess whether these potentials can discriminate the native conformation from the manifold of alternative, low-energy misfolded states. Nevertheless, these results also have significant implications for the development of a methodology for multiscale modeling of proteins that combines reduced and detailed atomic models. Proteins 1999;35:447-452. 1999 Wiley-Liss, Inc.

Proteins: Structure, Function, and Bioinformatics, 2010

The present study addresses the effect of structural distortion, caused by protein modeling error... more The present study addresses the effect of structural distortion, caused by protein modeling errors, on calculated binding affinities toward small molecules. The binding affinities to a total of 300 distorted structures based on five different protein-ligand complexes were evaluated to establish a broadly applicable relationship between errors in protein structure and errors in calculated binding affinities. Relatively accurate protein models (less than 2 A RMSD within the binding site) demonstrate a 14.78 (+/-7.5)% deviation in binding affinity from that calculated by using the corresponding crystal structure. For structures of 2-3 A, 3-4 A, and >4 A RMSD within the binding site, the error in calculated binding affinity increases to 20.8 (+/-5.98), 22.79 (+/-11.3), and 29.43 (+/-11.47)%, respectively. The results described here may be used in combination with other tools to evaluate the utility of modeled protein structures for drug development or other ligand-binding studies.

Molecular Simulation, 2000

Abstract As the field of theoretical biophysics begins to recognize systems of longer timescales ... more Abstract As the field of theoretical biophysics begins to recognize systems of longer timescales and larger magnitude, rapid approaches for investigating these systems are required. One promising simplification of the typical system of a solute surrounded by water is the use of implicit solvation models. The generalized Born implicit solvent offers a rapid approach for computing the electrostatic effects of bulk solvent without the explicit representation of water molecules. This report describes the parameterization of a generalized Born (GB) model for protein and nucleic acid structures. As a demonstration of the usefulness of this approach, the GB model is applied toward the discrimination of misfolded and properly folded protein structures. This study attempts to illustrate the potential of the GB model for molecular dynamics simulations over longer timescales as well as for screening large structural databases.

Journal of the American Chemical Society, 2004

Journal of Molecular Biology, 2009

Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the ... more Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only UDGs but also xanthine DNA glycosylases (XDGs). In addition, mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2 for XDG and UDG activities. Mutations at M57 (M57L) and H210 (H210G, H210M, and H210N), both of which are involved in interactions with the C2 carbonyl oxygen in uracil or xanthine, cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1, which corresponds to a position involved in base flipping. This mutation results in an activity profile resembling a human SMUG1-like enzyme as exemplified by the retention of UDG activity on mismatched base pairs and weak XDG activity. MD simulations indicate that M57L increases the flexibility of the motif 2 loop region and specifically A214, which may account for the reduced catalytic activity. G60Y completely abolishes XDG and UDG activity, which is consistent with a modeled structure in which G60Y blocks the entry of either xanthine or uracil to the base binding pocket. Most interestingly, a proline substitution at the G63 position switches the Gme SMUG1 enzyme to an exclusive UDG as demonstrated by the uniform excision of uracil in both double-stranded and single-stranded DNA and the complete loss of XDG activity. MD simulations indicate that a combination of a reduced free volume and altered flexibility in the active-site loops may underlie the dramatic effects of the G63P mutation on the activity profile of SMUG1. This study offers insights on the important role that modulation of conformational flexibility may play in defining specificity and catalytic efficiency.

Journal of Molecular Biology, 2002

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilizati... more Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.

Journal of Molecular Biology, 2011

Journal of Molecular Biology, 2012

The development of a molecular-level understanding of drug resistance through β-lactamase is crit... more The development of a molecular-level understanding of drug resistance through β-lactamase is critical not only in designing newer-generation antibacterial agents but also in providing insight into the evolutionary mechanisms of enzymes in general. In the present study, we have evaluated the effect of four drug resistance mutations (A42G, E104K, G238S, and M182T) on the cefotaximase activity of the TEM-1 β-lactamase. Using computational methods, including docking and molecular mechanics calculations, we have been able to correctly identify the relative order of catalytic activities associated with these four single point mutants. Further analyses suggest that the changes in catalytic efficiency for mutant enzymes are correlated to structural changes within the binding site. Based on the energetic and structural analyses of the wild-type and mutant enzymes, structural rearrangement is suggested as a mechanism of evolution of drug resistance through TEM β-lactamase. The present study not only provides molecular-level insight into the effect of four drug resistance mutations on the structure and function of the TEM β-lactamase but also establishes a foundation for a future molecular-level analysis of complete evolutionary trajectory for this class of enzymes.

Journal of Medicinal Chemistry, 2004

Knowledge-based potentials have been found useful in a variety of biophysical studies of macromol... more Knowledge-based potentials have been found useful in a variety of biophysical studies of macromolecules. Recently, it has also been shown in self-consistent studies that it is possible to extract quantities consistent with pair potentials from model structural databases. In this study, we attempt to extend the results obtained from these self-consistent studies toward the extraction of realistic pair potentials from the Protein Data Bank (PDB). The new method utilizes a clustering approach to define atom types within the PDB consistent with the optimal effective pairwise potential. The method has been integrated into the SMoG drug design package, resulting in an improved approach for the rapid and accurate estimation of binding affinities from structural information. Using this approach, it is possible to generate simple knowledge-based potentials that correlate (R = 0.61) with experimental binding affinities in a database of 118 diverse complexes. Furthermore, predictions performed on a random 1/3 of the database consistently show an average unsigned error of 1.5 log Ki units. It is also possible to generate specialized knowledge-based potentials, targeted to specific protein families. This approach is capable of generating potentials that correlate strongly with experimental binding affinities within these families (R = 0.8-0.9). Predictions on 1/3 of these family databases yield average unsigned errors ranging from 1.1 to 1.3 log Ki units. In summary, we describe a physically motivated approach to optimizing knowledge-based potentials for binding energy prediction that can be integrated into a variety of stages within a lead discovery protocol.

Communications in Computational Physics

Salt influences protein stability through electrostatic mechanisms as well as through nonpolar Ho... more Salt influences protein stability through electrostatic mechanisms as well as through nonpolar Hofmeister effects. In the present work, a continuum solvation based model is developed to explore the impact of salt on protein stability. This model relies on a traditional Poisson-Boltzmann (PB) term to describe the polar or electrostatic effects of salt, and a surface area dependent term containing a salt concentration dependent microscopic surface tension function to capture the non-polar Hofmeister effects. The model is first validated against a series of cold-shock protein variants whose salt-dependent protein fold stability profiles have been previously determined experimentally. The approach is then applied to HIV-1 protease in order to explain an experimentally observed enhancement in stability and activity at high (1M) NaCl concentration. The inclusion of the salt-dependent non-polar term brings the model into quantitative agreement with experiment, and provides the basis for fu...

Communications in Computational Physics

Recently, it was demonstrated that implicit solvent models were capable of generating stable B-fo... more Recently, it was demonstrated that implicit solvent models were capable of generating stable B-form DNA structures. Specifically, generalized Born (GB) implicit solvent models have improved regarding the solvation of conformational sampling of DNA [1,2]. Here, we examine the performance of the GBSW and GBMV models in CHARMM for characterizing base flipping free energy profiles of undamaged and damaged DNA bases. Umbrella sampling of the base flipping process was performed for the bases cytosine, uracil and xanthine. The umbrella sampling simulations were carried-out with both explicit (TIP3P) and implicit (GB) solvent in order to establish the impact of the solvent model on base flipping. Overall, base flipping potential of mean force (PMF) profiles generated with GB solvent resulted in a greater free energy difference of flipping than profiles generated with TIP3P. One of the significant differences between implicit and explicit solvent models is the approximation of solute-solvent...

Journal of Computational Chemistry

One of the anthrax receptors, tumor endothelial marker 8 (TEM8), is reported to be a potential an... more One of the anthrax receptors, tumor endothelial marker 8 (TEM8), is reported to be a potential anticancer target due to its over‐expression during tumor angiogenesis. To extend our BioLayer Interferometry study in PA‐TEM8 binding, we present a computational approach to reveal the role of an integral metal ion on receptor structure and binding thermodynamics. We estimated the interaction energy between PA and TEM8 using computer simulation. Consistent with experimental study, computational results indicate the metal ion in TEM8 contributes significantly to the binding affinity, and PA‐TEM8 binding is more favorable in the presence of Mg2+ than Ca2+. Further, computational analysis suggests that the differences in PA‐TEM8 binding affinity are comparable to the closely related integrin proteins. The conformation change, which linked to changes in activity of integrins, was not found in TEM8. In the present of Mg2+, TEM8 remains in a conformation analogous to an integrin open (high‐affinity) conformation. © 2017 Wiley Periodicals, Inc.

Scientific reports, Apr 11, 2017

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Fami... more Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed. Mutual Information analysis identifies a complexed correlated mutation network including a strong correlation in the EG doublet in motif 1 of family 4 UDGa and in the QD doublet in motif 1 of family 1 UNG. Conversion of EG doublet in family 4 Thermus thermophilus UDGa to QD doublet increases the catalytic efficiency by over one hundred-fold and seventeen-fold over the E41Q and G42D single mutation, respectively, rectifying the strong correlation in the doublet. Molecular dynamics simulations suggest that the correlated mutations in the doublet in motif 1 position the catalytic H155 in m...

Communications in Computational Physics

Recently, it was demonstrated that implicit solvent models were capable of generating stable B-fo... more Recently, it was demonstrated that implicit solvent models were capable of generating stable B-form DNA structures. Specifically, generalized Born (GB) implicit solvent models have improved regarding the solvation of conformational sampling of DNA [1, 2]. Here, we examine the performance of the GBSW and GBMV models in CHARMM for characterizing base flipping free energy profiles of undamaged and damaged DNA bases. Umbrella sampling of the base flipping process was performed for the bases cytosine, uracil and xanthine. The umbrella sampling simulations were carried-out with both explicit (TIP3P) and implicit (GB) solvent in order to establish the impact of the solvent model on base flipping. Overall, base flipping potential of mean force (PMF) profiles generated with GB solvent resulted in a greater free energy differ-ence of flipping than profiles generated with TIP3P. One of the significant differences between implicit and explicit solvent models is the approximation of solute-solve...

Nucleic acids research, 2015

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-s... more The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb...

The Journal of biological chemistry, Jan 9, 2011

DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigation... more DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigations identified five families within the uracil-DNA glycosylase (UDG) superfamily. All enzymes within the superfamily studied thus far exhibit uracil-DNA glycosylase activity. Here we identify a new class of DNA glycosylases in the UDG superfamily that lacks UDG activity. Instead, these enzymes act as hypoxanthine-DNA glycosylases in vitro and in vivo. Molecular modeling and structure-guided mutational analysis allowed us to identify a unique catalytic center in this class of DNA glycosylases. Based on unprecedented biochemical properties and phylogenetic analysis, we propose this new class of DNA repair glycosylases that exists in bacteria, archaea, and eukaryotes as family 6 and designate it as the hypoxanthine-DNA glycosylase family. This study demonstrates the structural evolvability that underlies substrate specificity and catalytic flexibility in the evolution of enzymatic function.

The Journal of biological chemistry, Jan 27, 2014

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that fa... more UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro. Genetic analysis indicates that family 5 UDGb can also act as an enzyme to remove uracil incorporated into DNA through the existence of dUTP in the nucleotide pool. Mutational analysis coupled with molecular modeling and molecular dynamics analysis reveals that although hydrogen bonding to O2 of uracil underlies the UDG activity in a dissociative fashion, Tth UDGb re...

The Journal of Physical Chemistry B, 1999

... This factor remains limiting and to date has hampered the quest for relevant studies of prote... more ... This factor remains limiting and to date has hampered the quest for relevant studies of protein folding and dynamics on microsecond time ... 7. One model that provides an approximate continuum solvent representation is the generalized Born (GB) model introduced by Still and co ...

Proteins: Structure, Function, and Genetics, 1999

A protocol for the rapid energetic analysis of protein-ligand complexes has been developed. This ... more A protocol for the rapid energetic analysis of protein-ligand complexes has been developed. This protocol involves the generation of protein-ligand complex ensembles followed by an analysis of the binding free energy components. We apply this methodology toward understanding the origin of binding specificity within the human immunodeficiency virus/feline immunodeficiency virus (HIV/FIV) protease system, a model system for drug resistance studies. A distinct difference in the internal strain of an inhibitor within each protein environment clearly favors the HIV protease complex, as observed experimentally. Our analysis also predicts that residues within the S2-S3 pockets of the FIV protease active site are responsible for this strain. Close examination of the active site residue contributions to interaction energy and desolvation energy identifies specific amino acids that may also play a role in determining the binding preferences of these two enzymes. Proteins 1999;36:318-331.

Proteins: Structure, Function, and Genetics, 1999

The relationship between the unfolding pseudo free energies of reduced and detailed atomic models... more The relationship between the unfolding pseudo free energies of reduced and detailed atomic models of the GCN4 leucine zipper is examined. Starting from the native crystal structure, a large number of conformations ranging from folded to unfolded were generated by all-atom molecular dynamics unfolding simulations in an aqueous environment at elevated temperatures. For the detailed atomic model, the pseudo free energies are obtained by combining the CHARMM all-atom potential with a solvation component from the generalized Born, surface accessibility, GB/SA, model. Reduced model energies were evaluated using a knowledge-based potential. Both energies are highly correlated. In addition, both show a good correlation with the root mean square deviation, RMSD, of the backbone from native. These results suggest that knowledge-based potentials are capable of describing at least some of the properties of the folded as well as the unfolded states of proteins, even though they are derived from a database of native protein structures. Since only conformations generated from an unfolding simulation are used, we cannot assess whether these potentials can discriminate the native conformation from the manifold of alternative, low-energy misfolded states. Nevertheless, these results also have significant implications for the development of a methodology for multiscale modeling of proteins that combines reduced and detailed atomic models. Proteins 1999;35:447-452. 1999 Wiley-Liss, Inc.

Proteins: Structure, Function, and Bioinformatics, 2010

The present study addresses the effect of structural distortion, caused by protein modeling error... more The present study addresses the effect of structural distortion, caused by protein modeling errors, on calculated binding affinities toward small molecules. The binding affinities to a total of 300 distorted structures based on five different protein-ligand complexes were evaluated to establish a broadly applicable relationship between errors in protein structure and errors in calculated binding affinities. Relatively accurate protein models (less than 2 A RMSD within the binding site) demonstrate a 14.78 (+/-7.5)% deviation in binding affinity from that calculated by using the corresponding crystal structure. For structures of 2-3 A, 3-4 A, and >4 A RMSD within the binding site, the error in calculated binding affinity increases to 20.8 (+/-5.98), 22.79 (+/-11.3), and 29.43 (+/-11.47)%, respectively. The results described here may be used in combination with other tools to evaluate the utility of modeled protein structures for drug development or other ligand-binding studies.

Molecular Simulation, 2000

Abstract As the field of theoretical biophysics begins to recognize systems of longer timescales ... more Abstract As the field of theoretical biophysics begins to recognize systems of longer timescales and larger magnitude, rapid approaches for investigating these systems are required. One promising simplification of the typical system of a solute surrounded by water is the use of implicit solvation models. The generalized Born implicit solvent offers a rapid approach for computing the electrostatic effects of bulk solvent without the explicit representation of water molecules. This report describes the parameterization of a generalized Born (GB) model for protein and nucleic acid structures. As a demonstration of the usefulness of this approach, the GB model is applied toward the discrimination of misfolded and properly folded protein structures. This study attempts to illustrate the potential of the GB model for molecular dynamics simulations over longer timescales as well as for screening large structural databases.

Journal of the American Chemical Society, 2004

Journal of Molecular Biology, 2009

Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the ... more Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only UDGs but also xanthine DNA glycosylases (XDGs). In addition, mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2 for XDG and UDG activities. Mutations at M57 (M57L) and H210 (H210G, H210M, and H210N), both of which are involved in interactions with the C2 carbonyl oxygen in uracil or xanthine, cause substantial reductions in XDG and UDG activities. Increased selectivity is achieved in the A214R mutant of Gme SMUG1, which corresponds to a position involved in base flipping. This mutation results in an activity profile resembling a human SMUG1-like enzyme as exemplified by the retention of UDG activity on mismatched base pairs and weak XDG activity. MD simulations indicate that M57L increases the flexibility of the motif 2 loop region and specifically A214, which may account for the reduced catalytic activity. G60Y completely abolishes XDG and UDG activity, which is consistent with a modeled structure in which G60Y blocks the entry of either xanthine or uracil to the base binding pocket. Most interestingly, a proline substitution at the G63 position switches the Gme SMUG1 enzyme to an exclusive UDG as demonstrated by the uniform excision of uracil in both double-stranded and single-stranded DNA and the complete loss of XDG activity. MD simulations indicate that a combination of a reduced free volume and altered flexibility in the active-site loops may underlie the dramatic effects of the G63P mutation on the activity profile of SMUG1. This study offers insights on the important role that modulation of conformational flexibility may play in defining specificity and catalytic efficiency.

Journal of Molecular Biology, 2002

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilizati... more Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.

Journal of Molecular Biology, 2011

Journal of Molecular Biology, 2012

The development of a molecular-level understanding of drug resistance through β-lactamase is crit... more The development of a molecular-level understanding of drug resistance through β-lactamase is critical not only in designing newer-generation antibacterial agents but also in providing insight into the evolutionary mechanisms of enzymes in general. In the present study, we have evaluated the effect of four drug resistance mutations (A42G, E104K, G238S, and M182T) on the cefotaximase activity of the TEM-1 β-lactamase. Using computational methods, including docking and molecular mechanics calculations, we have been able to correctly identify the relative order of catalytic activities associated with these four single point mutants. Further analyses suggest that the changes in catalytic efficiency for mutant enzymes are correlated to structural changes within the binding site. Based on the energetic and structural analyses of the wild-type and mutant enzymes, structural rearrangement is suggested as a mechanism of evolution of drug resistance through TEM β-lactamase. The present study not only provides molecular-level insight into the effect of four drug resistance mutations on the structure and function of the TEM β-lactamase but also establishes a foundation for a future molecular-level analysis of complete evolutionary trajectory for this class of enzymes.

Journal of Medicinal Chemistry, 2004

Knowledge-based potentials have been found useful in a variety of biophysical studies of macromol... more Knowledge-based potentials have been found useful in a variety of biophysical studies of macromolecules. Recently, it has also been shown in self-consistent studies that it is possible to extract quantities consistent with pair potentials from model structural databases. In this study, we attempt to extend the results obtained from these self-consistent studies toward the extraction of realistic pair potentials from the Protein Data Bank (PDB). The new method utilizes a clustering approach to define atom types within the PDB consistent with the optimal effective pairwise potential. The method has been integrated into the SMoG drug design package, resulting in an improved approach for the rapid and accurate estimation of binding affinities from structural information. Using this approach, it is possible to generate simple knowledge-based potentials that correlate (R = 0.61) with experimental binding affinities in a database of 118 diverse complexes. Furthermore, predictions performed on a random 1/3 of the database consistently show an average unsigned error of 1.5 log Ki units. It is also possible to generate specialized knowledge-based potentials, targeted to specific protein families. This approach is capable of generating potentials that correlate strongly with experimental binding affinities within these families (R = 0.8-0.9). Predictions on 1/3 of these family databases yield average unsigned errors ranging from 1.1 to 1.3 log Ki units. In summary, we describe a physically motivated approach to optimizing knowledge-based potentials for binding energy prediction that can be integrated into a variety of stages within a lead discovery protocol.