Abstracts of Articles in 1996

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1. The Low Dielectric Interior of Proteins is Sufficient to Cause Major Structural Changes in DNA on Association
2. Structural Fluctuations of a Cryptophane Host: A Molecular Dynamics Simulation
3. Surface Titration: A Continuum Electrostatics Model
4. Time-Dependent Rate Coefficients from Brownian Dynamics Simulations
5. Free Energy Simulations: Correcting for Electrostatic Cutoffs by Use of the Poisson Equation
6. The Determinants of pK_as in Proteins
7. Quantum-Classical Molecular Dynamics Simulations of Proton Transfer Processes in Molecular Complexes and in Enzymes
8. Orientational Steering in Enzyme-Substrate Association: Ionic Strength Dependence of Hydrodynamic Torque Effects
9. Comparison of Continuum and Explicit Models of Solvation: Potentials of Mean Force for Alanine Dipeptide
10. Binding of Tacrine and 6-Chlorotacrine by Acetylcholinesterase
11. Use of the Grand Canonical Ensemble in Potential of Mean Force Calculations
12. Study of Global Motions in Proteins by Weighted Masses Molecular Dynamics: Adenylate Kinase as a Test Case
13. Theoretical Study of Inhibition of Adenosine Deaminase by 8R-Coformycin and 8R-Deoxycoformycin
14. Density Functional Based Parametrization of a Valence Bond Method and its Applications in Quantum-Classical Molecular Dynamics Simulations of Enzymatic Reactions
15. Electrostatic Channeling in the Bifunctional Enzyme Dihydrofolate Reductase-Thymidylate Synthase
16. Computational Science: New Horizons and Relevance to Pharmaceutical Design
17. A Speed Limit for Protein Folding (Commentary)
18. Evidence for Electrostatic Channeling in a Fusion Protein of Malate Dehydrogenase and Citrate Synthase
19. Acetylcholinesterase: Role of the Enzyme's Charge Distribution in Steering Charged Ligands Toward the Active Site
20. Computing the Ionization States of Proteins with a Detailed Charge Model
21. A 240-Fold Electrostatic Rate-Enhancement for Acetylcholinesterase - Substrate Binding can be Predicted by the Potential within the Active Site

The Determinants of pK_as in ProteinsJan Antosiewicz, J. Andrew McCammon and Michael K. GilsonBiochemistry, Vol. 35, No. 24, pp. 7819-7833 (1996)    [PubMed 8672483]
The formulation of realistic and predictive models for protonation equilibria in proteins represents a fundamental challenge in physical chemistry. A number of laboratories have used detailed solutions of the linearized Poisson- Boltzmann (PB) equation for proteins in solution to estimate the energetics of ionization processes. Although certain components of the PB model, such as the ionic strength and the dielctric constant of the solvent, are fixed by the experimental conditions to be simulated, other components of the model are less certain. These components include the atomic charges and radii used in the electrostatics calculations; the conformation or conformational distribution of the protein; and the dielectric constant of the protein. The present study makes use of the substantial body of structural and pKa data for the proteins hen egg white lysozyme (HEWL), bovine pancreatic ribonuclease A (RNAse A), bovine pancreatic trypsin inhibitor (PTI), and turkey ovomucoid third domain (OMTKY3), to examine the influence of the dielectric constant of the protein, of atomic parameters, and of protein conformation, upon the accuracy of the calculations. This part of the paper is based upon cumulative comparisons of computed pKas with measurements, and aims to establish models that are increasingly accurate and thus, it is presumed, increasingly realistic. However, closer examination of some of these systems, and of the experimental data themselves, is also of considerable interest. Accordingly, an additional study of the influence of bound phosphate upon the pKas of histidine side chains in RNAse A is provided, and the basis for the pKa shifts or carboxylic acids in OMTKY3 is analyzed. In addition, observations resulting from a cumulative analysis of 63 measured and calculated pKas are presented.

Orientational Steering in Enzyme-Substrate Association: Ionic Strength Dependence of Hydrodynamic Torque EffectsJan Antosiewicz, James M. Briggs and J. Andrew McCammonEuropean Biophysics Journal, Vol. 24, No. 3, pp. 137-141 (1996)    [PubMed 8852560]
The effect of hydrodynamic torques on the association rate constants for enzyme-ligand complexation is investigated by Brownian dynamics simulations. Our hydrodynamic models of the enzyme and ligand are composed of spherical elements with friction forces acting at their centers. A quantitative measure of hydrodynamic torque orientational effects is introduced by choosing, as a reference system, an enzyme-ligand model with the same average hydrodynamic interactions but without orientational dependence. Our simple models show a 15% increase in the rate constant caused by hydrodynamic torques at physiological ionic strength. For more realistic hydrodynamic models, which are not computationally feasible at present, this effect is probably higher. The most important finding of this work is that hydrodynamic complementarity in shape (i.e. like the fitting together of pieces of a puzzle) is most effective for interactions between molecules at physiological ionic strength.

Binding of Tacrine and 6-Chlorotacrine by AcetylcholinesteraseS.T. Wlodek, J. Antosiewicz, J.A. McCammon, T.P. Straatsma, M.K. Gilson, J.M. Briggs, C. Humblet and J.L. SussmanBiopolymers, Vol. 38, Issue 1, pp. 109-117 (1996)    [PubMed 8679940]
Multiconfiguration thermodynamic integration was used to determine the relative binding strength of tacrine and 6-chlorotacrine by Torpedo californica acetylcholinesterase. 6-Chlorotacrine appears to be bound stronger by 0.7+/-0.4 kcal/mol than unsubstituted tacrine when the active site triad residue His-440 is deprotonated. This result is in excellent agreement with experimental inhibition data on electric eel acetylcholinesterase. Electrostatic Poisson-Boltzmann calculations confirm that order of binding strength, resulting in ΔG of binding of -2.9 and -3.3 kcal/mol for tacrine and chlorotacrine, respectively, and suggest inhibitor binding does not occur when His-440 is charged. Our results suggest that electron density redistribution upon tacrine chlorination is mainly responsible for the increased attraction potential between protonated inhibitor molecule and adjacent aromatic groups of Phe-330 and Trp-84.

Study of Global Motions in Proteins by Weighted Masses Molecular Dynamics: Adenylate Kinase as a Test CaseSamir Elamrani, Michael B. Berry, George N. Phillips, Jr. and J. Andrew McCammonProteins, Vol. 25, Issue 1, pp. 79-88 (1996)    [PubMed 8727320]
The weighted masses molecular dynamics (WMMD) technique is applied to the protein adenylate kinase. A novel set of restraints has been developed to allow the use of this technique with proteins. The WMMD simulation is successful in predicting the flexibility of the two mobile domains of the protein. The end product of the simulation is similar to the known open and AMP bound forms of the enzyme. The biological relevance of the restraints used and potential methods of improving the technique are discussed.

Theoretical Study of Inhibition of Adenosine Deaminase by 8R-Coformycin and 8R-DeoxycoformycinTami J. Marrone, T.P. Straatsma, James M. Briggs, David K. Wilson, Florante A. Quiocho and J. Andrew McCammonJournal of Medicinal Chemistry, Vol. 39, No. 1, pp. 277-284 (1996)    [PubMed 8568817]
Molecular dynamics and free energy simulations were performed to examine the binding of (8R)-deoxycoformycin and (8R)-coformycin to adenosine deaminase. The two inhibitors differ only at the 2' position of the sugar ring; the sugar moiety of conformycin is ribose, while it is deoxyribose for deoxycoformycin. The 100 ps molecular dynamics trajectories reveal that Asp 19 and His 17 interact strongly with the 5' hydroxyl group of the sugar moiety of both inhibitors and appear to play an important role in binding the sugar. The 2' and 3' groups of the sugars are near the protein-water interface and can be stabilized by either protein residues or water. The flexibility of the residues at the opening of the active site helps to explain the modest difference in binding of the two inhibitors and how substrates/inhibitors can enter an otherwise inaccessible binding site.

Electrostatic Channeling in the Bifunctional Enzyme Dihydrofolate Reductase-Thymidylate SynthaseAdrian H. Elcock, Michael J. Potter, David A. Matthews, Daniel R. Knighton and J. Andrew McCammonJournal of Molecular Biology, Vol. 262, Issue 3, pp. 370-374 (1996)    [PubMed 8845002]
The bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) carries out two distinct reactions, with the dihydrofolate produced by the TS-catalysed reaction acting as the substrate for the DHFR-catalysed reaction. Brownian dynamics simulation techniques were used to investigate the possible role of electrostatics in determining efficient channeling of the substrate, by explicitly simulating substrate diffusion between the two active sites. With a substrate charge of -2, almost all (>95%) substrate molecules leaving the TS active site reached the DHFR active site at zero ionic strength. Under the same conditions, but in the absence of electrostatic effects, successful channeling was reduced to only around 6%: electrostatic effects therefore appear essential to explain the efficient channeling observed experimentally. The importance of substrate charge, the relative contributions of specific basic residues in the protein, the role played by the second monomer of the dimer in channeling and the effects of changing ionic strength were all investigated. Simulations performed for substrate transfer in the opposite direction suggest that channeling in DHFR-TS is not strongly directional and that the role of electrostatics is perhaps more one of restricting diffusion of the substrate than one of actively guiding it from the TS to the DHFR active site. The results demonstrate that electrostatic channeling can be a highly efficient means of transferring charged substrates between active sites in solvent-exposed environments.

Computational Science: New Horizons and Relevance to Pharmaceutical DesignJames M. Briggs, Tami J. Marrone and J. Andrew McCammonTrends in Cardiovascular Medicine, Vol. 6, Issue 6, pp. 198-204 (1996)    [PubMed 21232297]
Computer methods are used extensively in the design and refinement of drug leads. A short summary is given for several computational methods followed by a description of how some of these methods have been applied to design drugs targeted to the renin-angiotensin system and to cholinergic synapses. These methods include quantitative structure activity relationship (QSAR) methods, comparative molecular field analyses (CoMFA), 3D database searching, de novo design of ligands, docking, and computational alchemy (free energy perturbation, FEP, thermodynamic integration, MCTI). Most of these methods can be used whether or not detailed structural information about the binding site is available, although without an X-ray structure, the analyses are more qualitative. All of these methods are used extensively in the commercial design of pharmaceuticals. The main problem with most of these mthods is in the scoring (ranking) of interactions or matches. Advances in this area and others (methods development and increase in capabilities of computers) will increase the predictive power of these mthods and help to speed the time to market of new pharmaceuticals.

A Speed Limit for Protein Folding (Commentary)J. Andrew McCammonProceedings of the National Academy of Sciences of the USA, Vol. 93, Issue 21, pp. 11426-11427 (1996)    [PubMed 8876151]
Recent observations of fast steps in protein folding address the question: What is the shortest time that would be required for folding? The paper by Hagen et al. in the current issue of the Proceedings provides strong experimental support for a speed limit that is governed by diffusion. Diffusion is known to limit the rates of many of the activities of proteins, including enzymatic activity, electron transfer, and binding to other macromolecules. The new evidence suggests that folding can be added to this list.

Evidence for Electrostatic Channeling in a Fusion Protein of Malate Dehydrogenase and Citrate SynthaseAdrian H. Elcock and J. Andrew McCammonBiochemistry, Vol. 35, No. 39, pp. 12652-12658 (1996)    [PubMed 8841108]
Brownian dynamics simulations were performed to investigate a possible role for electrostatic channeling in transferring substrate between two of the enzymes of the citric acid cycle. The diffusion of oxaloacetate from one of the active sites of malate dehydrogenase (MDH) to the active sites of citrate synthase (CS) was simulated in the presence and absence of electrostatic forces using a modeled structure for a MDH-CS fusion protein. In the absence of electrostatic forces, fewer than 1% of substrate molecules leaving the MDH active site are transferred to CS. When electrostatic forces are present at zero ionic stength however, around 45% of substrate molecules are successfully channeled. As expected for an electrostatic mechanism of transfer, increasing the ionic strength in the simulations reduces the calculated transfer efficiency. Even at 150mM however, the inclusion of electrostatic forces results in an increase in transfer efficiency of more than one order of magnitude. The simulations therefore provide evidence for the involvement of electrostatic channeling in guiding substrate transfer between two of the enzymes of the citric acid cycle. Similar effects may opeate between other members of the citric acid metabolon.

Acetylcholinesterase: Role of the Enzyme's Charge Distribution in Steering Charged Ligands Toward the Active SiteJan Antosiewicz, Stanislaw T. Wlodek and J. Andrew McCammonBiopolymers, Vol. 39, Issue 1, pp. 85-94 (1996)    [PubMed 8924629]
The electrostatic steering of charged ligands toward the active site of Torpedo californica acetylcholinesterase is investigated by Brownian dynamics simulations of wild type enzyme and several mutated forms, in which some normally charged residues are neutralized. The simulations reveal that the total ligand influx through a surface of 42A radius centered in the enzyme monomer and separated from the protein surface by 1-14A is not significantly influenced by electrostatic interactions. Electrostatic effects are visible for encounters with a surface of 32A radius, which is partially hidden inside the protein, but mostly within the solvent. A clear accumulation of encounter events for that sphere is observed in the area directly above the entrance to the active site gorge. In this area, the encounter events are increased by 40% compared to the case of a netural ligand, However, the differences among the encounter rates for the various mutants considered here are not pronouned, all rate constants being within plus or minus 10% of the average value. The enzyme charge distribution becomes more important as the charged ligand moves toward the bottom of the gorge, where the active site is located. We show that neither the enzyme's total charge, nor its dipole moment, fully account for the electrostatic steering of ligand to the active site. Higher moments of the enzyme's charge distribution are also important. However, for a series of mutations for which the direction of the enzyme dipole moment is constant within a few degrees, one observes a gradual decrease in the diffusional encounter rate constant with the number of neutralized residues. On the other hand, for other mutants that change the direction of the dipole moment from that of the wild type, the calculated encounter rate constants can be very close to that of the wild type. The present work yields two new insights to the kinetics of acetylcholinesterase. First, evolution appears to have built a redundant electrostatic steering capability into this important enzyme through the overall distribution of its thousands of partially charged atoms. And second, roughly half of the rate enhancement due to electrostatics arises from steering of the substrate outside the enzyme; the other half of the rate enhancement arises from improved trapping of the substrate after it had entered the gorge. The computational results reproduce qualitatively, and help to rationalize, many surprising experimental results obtained recently for human acetylcholinesterase.