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Arieh Warshel

Publications and source records attributed to Arieh Warshel.

At least 19 recordsLinked to original sources
PubMed2007

Modifying the beta,gamma leaving-group bridging oxygen alters nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA polymerase beta.

DNA polymerase catalysis and fidelity studies typically compare incorporation of "right" versus "wrong" nucleotide bases where the leaving group is pyrophosphate. Here we use dGTP analogues replacing the beta,gamma-bridging O with CH2, CHF, CF2, or CCl2 to explore leaving-group effects on the nucleotidyl transfer mechanism and fidelity of DNA polymerase (pol) beta. T.G mismatches occur with fidelities similar to dGTP with the exception of the CH2 analogue, which is incorporated with 5-fold higher fidelity. All analogues are observed to bind opposite template C with Kds between 1 and 4 microM, and structural evidence suggests that the analogues bind in essentially the native conformation, making them suitable substrates for probing linear free energy relationships (LFERs) in transient-kinetics experiments. Importantly, Brnsted correlations of log(kpol) versus leaving-group pKa for both right and wrong base incorporation reveal similar sensitivities (betalg approximately -0.8) followed by departures from linearity, suggesting that a chemical step rather than enzyme conformational change is rate-limiting for either process. The location of the breaks relative to pKas of CF2, O, and the sterically bulky CCl2-bridging compounds suggests a modification-induced change in the mechanism by stabilization of leaving-group elimination. The results are addressed theoretically in terms of the energetics of successive primer 3'-O addition (bond forming) and pyrophosphate analogue elimination (bond breaking) reaction energy barriers.

Base Sequence↗
PubMed2006

On the mechanism of hydrolysis of phosphate monoesters dianions in solutions and proteins.

The nature of the hydrolysis of phosphate monoester dianions in solutions and in proteins is a problem of significant current interest. The present work explores this problem by systematic calculations of the potential surfaces of the reactions of a series of phosphate monoesters with different leaving groups. These calculations involve computational studies ranging from ab initio calculations with implicit solvent models to ab initio QM/MM free energy calculations. The calculations reproduce the observed linear free energy relationship (LFER) for the solution reaction and thus are consistent with the overall experimental trend and can be used to explore the nature of the transition state (TS) region, which is not accessible to direct experimental studies. It is found that the potential surface for the associative and dissociative paths is very flat and that the relative height of the associative and dissociative TS is different in different systems. In general, the character of the TS changes from associative to dissociative upon decrease in the pKa of the leaving group. It is also demonstrated that traditional experimental markers such as isotope effects and the LFER slope cannot be used in a conclusive way to distinguish between the two classes of transition states. In addition it is found that the effective charges of the TS do not follow the previously assumed simple rule. Armed with that experience we explore the free energy surface for the GTPase reaction of the RasGap system. In this case it is found that the surface is flat but that the lowest TS is associative. The present study indicates that the nature of the potential surfaces for the phosphoryl transfer reactions in solution and proteins is quite complicated and cannot be determined in a conclusive way without the use of careful theoretical studies that should, of course, reproduce the available experimental information.

GTPase-Activating Proteins↗
PubMed2006

Magnesium-cationic dummy atom molecules enhance representation of DNA polymerase beta in molecular dynamics simulations: improved accuracy in studies of structural features and mutational effects.

Human DNA polymerase beta (pol beta) fills gaps in DNA as part of base excision DNA repair. Due to its small size it is a convenient model enzyme for other DNA polymerases. Its active site contains two Mg(2+) ions, of which one binds an incoming dNTP and one catalyzes its condensation with the DNA primer strand. Simulating such binuclear metalloenzymes accurately but computationally efficiently is a challenging task. Here, we present a magnesium-cationic dummy atom approach that can easily be implemented in molecular mechanical force fields such as the ENZYMIX or the AMBER force fields. All properties investigated here, namely, structure and energetics of both Michaelis complexes and transition state (TS) complexes were represented more accurately using the magnesium-cationic dummy atom model than using the traditional one-atom representation for Mg(2+) ions. The improved agreement between calculated free energies of binding of TS models to different pol beta variants and the experimentally determined activation free energies indicates that this model will be useful in studying mutational effects on catalytic efficiency and fidelity of DNA polymerases. The model should also have broad applicability to the modeling of other magnesium-containing proteins.

Binding Sites↗
PubMed2006

Using the constrained DFT approach in generating diabatic surfaces and off diagonal empirical valence bond terms for modeling reactions in condensed phases.

The empirical valence bond (EVB) model provides an extremely powerful way for modeling and analyzing chemical reactions in solutions and proteins. However, this model is based on the unverified assumption that the off diagonal elements of the EVB Hamiltonian do not change significantly upon transfer of the reacting system from one phase to another. This ad hoc assumption has been rationalized by its consistency with empirically observed linear free energy relationships, as well as by other qualitative considerations. Nevertheless, this assumption has not been rigorously established. The present work explores the validity of the above EVB key assumption by a rigorous numerical approach. This is done by exploiting the ability of the frozen density functional theory (FDFT) and the constrained density functional theory (CDFT) models to generate convenient diabatic states for QM/MM treatments, and thus to examine the relationship between the diabatic and adiabatic surfaces, as well as the corresponding effective off diagonal elements. It is found that, at least for the test case of S(N)()2 reactions, the off diagonal element does not change significantly upon moving from the gas phase to solutions and thus the EVB assumption is valid and extremely useful.

Algorithms↗
PubMed2006

The barrier for proton transport in aquaporins as a challenge for electrostatic models: the role of protein relaxation in mutational calculations.

The origin of the barrier for proton transport through the aquaporin channel is a problem of general interest. It is becoming increasingly clear that this barrier is not attributable to the orientation of the water molecules across the channel but rather to the electrostatic penalty for moving the proton charge to the center of the channel. However, the reason for the high electrostatic barrier is still rather controversial. It has been argued by some workers that the barrier is due to the so-called NPA motif and/or to the helix macrodipole or to other specific elements. However, our works indicated that the main reason for the high barrier is the loss of the generalized solvation upon moving the proton charge from the bulk to the center of the channel and that this does not reflect a specific repulsive electrostatic interaction but the absence of sufficient electrostatic stabilization. At this stage it seems that the elucidation and clarification of the origin of the electrostatic barrier can serve as an instructive test case for electrostatic models. Thus, we reexamine the free-energy surface for proton transport in aquaporins using the microscopic free-energy perturbation/umbrella sampling (FEP/US) and the empirical valence bond/umbrella sampling (EVB/US) methods as well as the semimacroscopic protein dipole Langevin dipole model in its linear response approximation version (the PDLD/S-LRA). These extensive studies help to clarify the nature of the barrier and to establish the "reduced solvation effect" as the primary source of this barrier. That is, it is found that the barrier is associated with the loss of the generalized solvation energy (which includes of course all electrostatic effects) upon moving the proton charge from the bulk solvent to the center of the channel. It is also demonstrated that the residues in the NPA region and the helix dipole cannot be considered as the main reasons for the electrostatic barrier. Furthermore, our microscopic and semimacroscopic studies clarify the problems with incomplete alternative calculations, illustrating that the effects of various electrostatic elements are drastically overestimated by macroscopic calculations that use a low dielectric constant and do not consider the protein reorganization. Similarly, it is pointed out that microscopic potential of mean force calculations that do not evaluate the electrostatic barrier relative to the bulk water cannot be used to establish the origin of the electrostatic barrier. The relationship between the present study and calculations of pK(a)s in protein interiors is clarified, pointing out that approaches that are applied to study the aquaporin barrier should be validated by pK(a)s calculations. Such calculations also help to clarify the crucial role of solvation energies in establishing the barrier in aquaporins.

Aquaporins↗
PubMed2006

Transition state theory can be used in studies of enzyme catalysis: lessons from simulations of tunnelling and dynamical effects in lipoxygenase and other systems.

The idea that enzyme catalysis involves special factors such as coherent fluctuations, quantum mechanical tunnelling and non-equilibrium solvation (NES) effects has gained popularity in recent years. It has also been suggested that transition state theory (TST) cannot be used in studies of enzyme catalysis. The present work uses reliable state of the art simulation approaches to examine the above ideas. We start by demonstrating that we are able to simulate any of the present catalytic proposals using the empirical valence bond (EVB) potential energy surfaces, the dispersed polaron model and the quantized classical path (QCP) approach, as well as the approximate vibronic method. These approaches do not treat the catalytic effects by phenomenological treatments and thus can be considered as first principles approaches (at least their ability to compare enzymatic reaction to the corresponding solution reactions). This work will consider the lipoxygenase reaction, and to lesser extent other enzymes, for specific demonstration. It will be pointed out that our study of the lipoxygenase reaction reproduces the very large observed isotope effect and the observed rate constant while obtaining no catalytic contribution from nuclear quantum mechanical (NQM) effects. Furthermore, it will be clarified that our studies established that the NQM effect decreases rather than increases when the donor-acceptor distance is compressed. The consequences of these findings in terms of the temperature dependence of the kinetic isotope effect and in terms of different catalytic proposals will be discussed. This paper will also consider briefly the dynamical effects and conclude that such effects do not contribute in a significant way to enzyme catalysis. Furthermore, it will be pointed out that, in contrast to recent suggestions, NES effects are not dynamical effects and should therefore be part of the activation free energy rather than the transmission factor. In view of findings of the present work and our earlier works, it seems that TST provides a quantitative tool for studies of enzyme catalysis and that the key open questions are related to the nature of the factors that lead to transition state stabilization.

Catalysis↗
PubMed2006

Modeling electrostatic effects in proteins.

Electrostatic energies provide what is perhaps the most effective tool for structure-function correlation of biological molecules. This review considers the current state of simulations of electrostatic energies in macromolecules as well as the early developments of this field. We focus on the relationship between microscopic and macroscopic models, considering the convergence problems of the microscopic models and the fact that the dielectric 'constants' in semimacroscopic models depend on the definition and the specific treatment. The advances and the challenges in the field are illustrated considering a wide range of functional properties including pK(a)'s, redox potentials, ion and proton channels, enzyme catalysis, ligand binding and protein stability. We conclude by pointing out that, despite the current problems and the significant misunderstandings in the field, there is an overall progress that should lead eventually to quantitative descriptions of electrostatic effects in proteins and thus to quantitative descriptions of the function of proteins.

Models, Molecular↗
PubMed2006

Using a charging coordinate in studies of ionization induced partial unfolding.

The ionization of groups in proteins may sometimes involve a partial unfolding and/or water penetration. Unfortunately the corresponding structural changes might not be captured by microscopic free energy perturbation (FEP) approaches due to activation barriers that are not surmounted in nanosecond FEP simulations. This problem is apparent, for example, from mutation experiments that introduced ionizable groups in protein interiors and from the difficulties to reproduce the corresponding pKa changes by microscopic approaches. Here we develop a new general approach for treating such challenging cases. Our approach drives the protein structural change by increasing the charge of the ionized group beyond its physical value and thus overcoming the barriers for the partial unfolding by a physically consistent process. The potential of our approach is illustrated by the evaluation of the pKa of the Val66Glu mutant of staphylococcal nuclease. In this case it is first demonstrated that standard FEP approaches give extremely disappointing results for this pKa. On the other hand, our "overcharging" approach gives a much more realistic result. We believe that the present approach represents a breakthrough in FEP studies of ionizable residues in proteins, and expect this strategy to be useful in studies of a wide range of challenging problems including simulations of hydrogen exchange processes.

Hydrogen↗
PubMed2006

Simulating the effect of DNA polymerase mutations on transition-state energetics and fidelity: evaluating amino acid group contribution and allosteric coupling for ionized residues in human pol beta.

The control of the catalytic power and fidelity of DNA polymerases involves the complex combined effect of the protein residues, the Mg2+ ions, and the interaction between the DNA bases. In an attempt to advance the understanding of catalytic control, we analyze the effect of the protein residues, taking human DNA polymerase beta as a model system. Specifically, we examine the ability of different theoretical models to reproduce the effect of ionized residues on the transition state (TS) binding energy and the corresponding k(pol)/KD. We also explore the role of the Mg2+ ions in the binding and catalysis processes. The application of the microscopic linear response approximation (LRA) and the semimacroscopic PDLD/S-LRA methods to a benchmark of mutational studies produces a semiquantitative correlation and indicates that these methods can provide predictive power. However, pre-steady-state and steady-state kinetic studies currently available do not give a unique benchmark, owing principally to widely varying experimental conditions. We believe that a more uniform experimental benchmark is needed for further refinement of the theoretical models. The analysis of the correlation between the results obtained by a rigorous thermodynamic cycle and by simpler approximations indicates that the protein reorganization between the open, i.e., unbound, form and the closed form does not change the magnitude of the calculated mutational effects in a major way for the experimental data used in this study. The use of the PDLD/S-LRA group contributions allows us to construct energy-based correlation diagrams that can help toward understanding the coupling, i.e., transfer of information, between the base-binding and catalytic sites and to gain a deeper insight into the molecular basis of DNA replication fidelity. Our analysis suggests that the allosteric matrix obtained by subtracting the correlation matrix of the correct and incorrect base pairs should prove useful in exploring the information transfer occurring between the base-binding and catalytic sites. This type of treatment should be especially effective when coupled with structural studies of polymerase-DNA-base mispair ternary complexes and studies using polymerase double mutants. We discuss the potential of direct calculations of binding energy of the TS in a rational design of TS analogues and in drug design.

Allosteric Regulation↗
PubMed2006

Advances in methods and algorithms in a modern quantum chemistry program package.

Advances in theory and algorithms for electronic structure calculations must be incorporated into program packages to enable them to become routinely used by the broader chemical community. This work reviews advances made over the past five years or so that constitute the major improvements contained in a new release of the Q-Chem quantum chemistry package, together with illustrative timings and applications. Specific developments discussed include fast methods for density functional theory calculations, linear scaling evaluation of energies, NMR chemical shifts and electric properties, fast auxiliary basis function methods for correlated energies and gradients, equation-of-motion coupled cluster methods for ground and excited states, geminal wavefunctions, embedding methods and techniques for exploring potential energy surfaces.

Algorithms↗
PubMed2006

Monte Carlo simulations of proton pumps: on the working principles of the biological valve that controls proton pumping in cytochrome c oxidase.

Gaining a detailed understanding of the proton-pumping process in cytochrome c oxidase (COX) is one of the challenges of modern biophysics. Recent mutation experiments have highlighted this challenge by showing that a single mutation (the N139D mutation) blocks the overall pumping while continuing to channel protons to the binuclear center without inhibiting the oxidase activity. Rationalizing this result has been a major problem because the mutation is quite far from E286, which is believed to serve as the branching point for the proton transport in the pumping process. In the absence of a reasonable explanation for this important observation, we have developed a Monte Carlo simulation method that can convert mutation and structural information to pathways for proton translocation and simulate the pumping process in COX on a millisecond and even subsecond time scale. This tool allows us to reproduce and propose a possible explanation to the effect of the N139D mutation and to offer a consistent model for the origin of the "valve effect" in COX, which is crucial for maintaining uphill proton pumping. Furthermore, obtaining the first structure-based simulation of proton pumping in COX, or in any other protein, indicates that our approach should provide a powerful tool for verification of mechanistic hypotheses about the action of proton transport proteins.

Biophysical Phenomena↗
PubMed2006

Towards accurate ab initio QM/MM calculations of free-energy profiles of enzymatic reactions.

Reliable studies of enzymatic reactions by combined quantum mechanical/molecular mechanics (QM/MM) approaches, with an ab initio description of the quantum region, presents a major challenge to computational chemists. The main problem is the need for a large amount of computer time to evaluate the QM energy, which in turn makes it extremely challenging to perform proper configurational sampling. This work presents major progress toward the evaluation of ab initio QM/MM free-energy surfaces and activation free energies of reactions in enzymes and in solutions. This is done by exploiting our previous idea of using the empirical valence bond (EVB) method as a reference potential and then using the linear response approximation (LRA) approach to evaluate the free energies of transfer from the EVB to the QM/MM surfaces in the reactant and product state. However, the new crucial step involves the use of a constraint at the transition state that fixes the system at a given value of the reaction coordinate and allows us to use the LRA at the transition state. The advance offered by the present approach is particularly significant because it evaluates the free energy associated with both the substrate and the solvent motions. This evaluation appeared to be a relatively simple task once one uses a classical reference potential. The main problem has been using the reference potential for the evaluation of the free-energy contributions associated with the solute motions where the difference between the reference EVB potential and the QM/MM potential can be large. The present refinement finally allows us to overcome the problems with the solute fluctuations and therefore to obtain, for the first time, a free-energy barrier that reflects the solute entropy properly. Thus, we present a way to evaluate the complete QM/MM activation free energy with an equal footing treatment of the solute and the solvent. This provides a general consistent and effective strategy for evaluating the QM/MM activation free energies in proteins and in solution. Our advance allows one to explore consistently various mechanistic and catalytic proposals while using ab initio (ai) QM/MM approaches.

Computer Simulation↗
PubMed2005

Through the channel and around the channel: Validating and comparing microscopic approaches for the evaluation of free energy profiles for ion penetration through ion channels.

Microscopic calculations of free energy profiles for ion transport through biological ion channels present a very serious challenge to modern simulation approaches. The main problem is due to the major convergence problems associated with the heterogeneous landscape of the electrostatic environment in ion channels and with the need to evaluate the profile associated with the transfer of the ion from bulk water to the channel environment. This problem is compounded by the lack of reliable and relevant benchmarks that can discriminate between alternative approaches. The present study is aimed at reducing the above problems by defining benchmarks that are directly relevant to ion channels and can also give converging results. This is done by constructing a series of models of a truncated gramicidin channel with different numbers of water molecules and by comparing the profiles for going around the channel and through the channel. These discriminating models are then used to validate and compare the adiabatic charging free energy perturbation (FEP) approach combined with an umbrella sampling approach (Warshel, A. J. Phys. Chem. 1982, 86, 2218) and the potential of mean force (PMF) approach used frequently in studies of ion channels. It is found that both approaches work quite well until one moves to the case of the fully solvated channel. In this limit, the PMF approach may give different results for the overall work of going through the channel and around the channel, while the FEP approach gives physically consistent results. The present benchmark also indicates that the weighted histogram analysis method (WHAM) approach does not offer a significant advantage over earlier approaches at least as much as studies of ion channels are concerned. Finally, it is concluded that the FEP approach may be more useful in evaluating the overall barrier for moving ions from water to ion channels and that in some cases it might be beneficial to use the FEP approach for selective points along the channel and then to connect these points by PMF calculations.

Algorithms↗
PubMed2005

What are the roles of substrate-assisted catalysis and proximity effects in peptide bond formation by the ribosome?

The action of the peptidyl transferase center of the large ribosomal unit presents a fundamental step in the evolution from the RNA world to the protein world. Thus, it is important to understand the origin of the catalytic power of this ancient enzyme. Earlier studies suggested that the ribosome catalyzes peptide bond formation by using one of its groups as a general base, while more recent works have proposed that the catalysis is due to proximity effects or to substrate-assisted catalysis. However, the actual nature of the catalytic mechanism remains controversial. This work addresses the origin of the catalytic power of the ribosome by using computer simulation approaches and comparing the energetics of the peptide bond formation in the ribosome and in water. It is found that a significant part of the observed activation entropy of the reference solution reaction is due to solvation entropy, and that the proximity effect is smaller than previously thought. It is also found that the 2'-OH of the A76 ribose, which is associated with a large rate acceleration in the ribosome reaction, does not catalyze peptide bond formation in water. Thus, the catalytic effect cannot be attributed to substrate-assisted catalysis but rather to the effect of the ribosome on the reacting system. Overall, our calculations indicate that the reduction of the activation free energy is mainly due to electrostatic effects. The nature of these effects and their relationship to catalytic factors in modern enzymes is analyzed and discussed.

Binding Sites↗
PubMed2005

On possible pitfalls in ab initio quantum mechanics/molecular mechanics minimization approaches for studies of enzymatic reactions.

Reliable studies of enzymatic reactions by combined quantum mechanics/molecular mechanics (QM/MM) approaches, with an ab initio description of the quantum region, presents a major challenge to computational chemists. The main problem is the need for a very large computer time for the evaluation of the QM energy, which in turn makes it extremely challenging to perform proper configurational sampling. A seemingly reasonable alternative is to perform energy minimization studies of the type used in gas-phase ab initio studies. However, it is hard to see why such an approach should give reliable results in protein active sites. To examine the problems with energy minimization QM/MM approaches, we chose the hypothetical reaction of a metaphosphate ion with water in the Ras.GAP complex. This hypothetical reaction served as a simple benchmark reaction. The possible problems with the QM/MM minimization were explored by generating several protein configurations from long MD simulations and using energy minimization and scanning of the reaction coordinates to evaluate the corresponding potential energy surfaces of the reaction for each of these different protein configurations. Comparing these potential energy surfaces, we found major variations of the corresponding minima. Furthermore, the reaction energies and activation energies also varied significantly even for similar protein configurations. The specific coordination of a magnesium ion, present in the active center of the protein complex, turned out to influence the energetics of the reaction in a major way, where a direct coordination to the reactant leads to an increase of the activation energy by 17 kcal/mol. Apparently, using energy minimization to generate potential surfaces for an enzymatic reaction, while starting from a single protein structure, could lead to major errors in calculations of activation free energies and binding free energies. Thus we believe that extensive samplings of the configurational space of the protein are essential for meaningful determination of the energetics of enzymatic reactions. The possible relevance of our conclusion with regard to a recent study of the RasGAP reaction is discussed.

Computational Biology↗
PubMed2005

Computer simulations of protein functions: searching for the molecular origin of the replication fidelity of DNA polymerases.

The use of computers to simulate the functions of complex biological macromolecules is essential to achieve a microscopic description of biological processes and to model and interpret experimental data. Here we apply theoretical computational approaches to investigate the fidelity of T7 DNA polymerase, divided into discrete steps that include contributions from substrate binding, pK(a) shifts, and rate constants for the PO bond-breaking and bond-making processes. We begin by defining the discrimination between right and wrong nucleotides in terms of the free energy landscape for the dNMP incorporation reaction. We then use the linear response approximation and the empirical valence bond methods to obtain converging results for the contribution of the binding and chemical steps to the overall fidelity. These approaches are successful in reproducing general trends in the observed polymerase incorporation fidelity. The calculations demonstrate the potential for further integration of theoretical and experimental studies to analyze high- and low-fidelity DNA polymerases.

Base Pairing↗