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Biomedical subjects

M K Gilson

Publications and source records attributed to M K Gilson.

At least 19 recordsLinked to original sources
PubMed2001

Interpreting trends in the binding of cyclic ureas to HIV-1 protease.

The design of new HIV protease inhibitors requires an improved understanding of the physical basis of inhibitor/protein binding. Here, the binding affinities of seven aliphatic cyclic ureas to HIV-1 protease are calculated using a predominant states method and an implicit solvent model based upon finite difference solutions of the Poisson-Boltzmann equation. The calculations are able to reproduce the observed U-shaped trend of binding free energy as a function of aliphatic chain length. Interestingly, the decrease in affinity for the longest chains is attributable primarily to the energy cost of partly desolvating charged aspartic and arginine groups at the mouths of the active site. Even aliphatic chains too short to contact these charged groups directly are subject to considerable desolvation penalties. We are not aware of other systems where binding affinity trends have been attributed to long-ranged electrostatic desolvation of ionized groups. A generalized Born/surface area solvation model yields a much smaller change in desolvation energy with chain length and, therefore, does not reproduce the experimental binding affinity trends. This result suggests that the generalized Born model should be used with caution for complex, partly desolvated systems like protein binding sites. We also find that changing the assumed protonation state of the active site aspartyl dyad significantly affects the computed binding affinity trends. The protonation state of the aspartyl dyad in the presence of cyclic ureas is discussed in light of the observation that the monoprotonated state reproduces the experimental results best.

Arginine↗
PubMed2001

The physical basis of nucleic acid base stacking in water.

It has been argued that the stacking of adenyl groups in water must be driven primarily by electrostatic interactions, based upon NMR data showing stacking for two adenyl groups joined by a 3-atom linker but not for two naphthyl groups joined by the same linker. In contrast, theoretical work has suggested that adenine stacking is driven primarily by nonelectrostatic forces, and that electrostatic interactions actually produce a net repulsion between adenines stacking in water. The present study provides evidence that the experimental data for the 3-atom-linked bis-adenyl and bis-naphthyl compounds are consistent with the theory indicating that nonelectrostatic interactions drive adenine stacking. First, a theoretical conformational analysis is found to reproduce the observed ranking of the stacking tendencies of the compounds studied experimentally. A geometric analysis identifies two possible reasons, other than stronger electrostatic interactions, why the 3-atom-linked bis-adenyl compounds should stack more than the bis-naphthyl compounds. First, stacked naphthyl groups tend to lie further apart than stacked adenyl groups, based upon both quantum calculations and crystal structures. This may prevent the bis-naphthyl compound from stacking as extensively as the bis-adenyl compound. Second, geometric analysis shows that more stacked conformations are sterically accessible to the bis-adenyl compound than to the bis-naphthyl compound because the linker is attached to the sides of the adenyl groups, but to the ends of the naphthyl groups. Finally, ab initio quantum mechanics calculations and energy decompositions for relevant conformations of adenine and naphthalene dimers support the view that stacking in these compounds is driven primarily by nonelectrostatic interactions. The present analysis illustrates the importance of considering all aspects of a molecular system when interpreting experimental data, and the value of computer models as an adjunct to chemical intuition.

Adenine↗
PubMed2001

Ligand-receptor docking with the Mining Minima optimizer.

The optimizer developed for the Mining Minima algorithm, which uses ideas from Genetic Algorithms, the Global Underestimator Method, and Poling, has been adapted for use in ligand-receptor docking. The present study describes the resulting methodology and evaluates its accuracy and speed for 27 test systems. The performance of the new docking algorithm appears to be competitive with that of previously published methods. The energy model, an empirical force field with a distance-dependent dielectric treatment of solvation, is adequate for a number of test cases, although incorrect low-energy conformations begin to compete with the correct conformation for larger sampling volumes and for highly solvent-exposed binding sites that impose little steric constraint on the ligand.

Algorithms↗
PubMed2001

BindingDB: a web-accessible molecular recognition database.

This paper presents an initial description of the BindingDB, a public web-accessible database of measured binding affinities for various molecular types (http://www.bindingdb.org). The BindingDB allows queries based upon a range of criteria, including chemical similarity or substructure, sequence homology, numerical criteria (e.g. delta G(o) < 5 kcal/mol) and reactant names (e.g. "lysozyme"). Principles of Human-Computer Interactions are being employed in creating the query interface and user-feedback is being solicited. The data specification includes significant experimental detail. A full dictionary has been created for isothermal titration calorimetry data in consultation with experimentalists and data dictionaries for enzyme-inhibition and other measurement techniques are being developed. Currently, the BindingDB contains several data sets of broad interest, such as antigen-antibody binding and cyclodextrin/small molecule binding. However, it is anticipated that online deposition by experimentalists will ultimately contribute to a larger flow of data. We are actively developing software and file specifications to facilitate such deposition.

Binding Sites↗
PubMed1999

Nucleic acid base-pairing and N-methylacetamide self-association in chloroform: affinity and conformation.

A recently developed computational method, 'mining minima', is used to examine the hydrogen-bonding interactions of nucleic acid base-pairs and of the N-methylacetamide homodimer in chloroform. The mining minima algorithm aggressively samples molecular conformations, identifies the most important local minima, and computes their contributions to the overall free energy of the system. Here, the CHARMM 98 parameter set is used for the potential energy and the generalized Born/surface area solvent model is used to account for the influence of the solvent. Good agreement with experiment is obtained for the non-covalent binding affinities of a series of complexes. The computational approach used here is applicable to a range of molecular systems.

Acetamides↗
PubMed1999

Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease.

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.

HIV Protease Inhibitors↗
PubMed1998

A hierarchical method for generating low-energy conformers of a protein-ligand complex.

A novel dynamical protocol for finding the low-energy conformations of a protein-ligand complex is described. The energy functions examined consist of an empirical force field with four different dielectric screening models; the generalized Born/surface area model also is examined. Application of the method to three complexes of known crystal structure provides insights into the energy functions used for selecting low-energy docked conformations and into the structure of the binding-energy surface. Evidence is presented that the local energy minima of a ligand in a binding site are arranged in a hierarchical fashion. This observation motivates the construction of a hierarchical docking algorithm that substantially enriches the population of ligand conformations close to the crystal conformation. The algorithm is also adapted to permit docking into a flexible binding site and preliminary tests of this method are presented.

Algorithms↗
PubMed1998

Theoretical and experimental analysis of ionization equilibria in ovomucoid third domain.

2D-NMR experiments were used to determine the pKa values ranging from 8.0 to >/=11.1 of seven basic residues in turkey ovomucoid third domain (OMTKY3) and were compared to values predicted as described by Antosiewicz et al. [(1996) Biochemistry 35, 7819-7833]. Lys 13, 29, and 34 were previously attributed with increasing the acidity of numerous acidic residues [Schaller, W., and Robertson, A. D. (1995) Biochemistry 34, 4714-4723]. These interactions were expected to raise the pKa values of those basic groups; however, the pKa values of Lys 13 and 34 are less than the model compound values. The pKa values of the other basic residues are greater than the model compound values and, unlike the acidic residues, all are surprisingly insensitive to salt. While the calculations properly predict the direction of most of the pKa shifts and provide valuable insight into the possible molecular origins of the interactions that perturb pKa values, there is a tendency to overestimate the magnitude of the shifts and their salt dependence. Interestingly, the shapes of both the calculated and observed transitions are often more complex than expected for a simple titration, suggesting that pKa values at many sites are changing during the transition. Differences between predicted and experimental pKa values and titration profiles for some residues may be due to as yet uncharacterized structural changes at the extremes of pH.

Models, Molecular↗
PubMed1997

The statistical-thermodynamic basis for computation of binding affinities: a critical review.

Although the statistical thermodynamics of noncovalent binding has been considered in a number of theoretical papers, few methods of computing binding affinities are derived explicitly from this underlying theory. This has contributed to uncertainty and controversy in certain areas. This article therefore reviews and extends the connections of some important computational methods with the underlying statistical thermodynamics. A derivation of the standard free energy of binding forms the basis of this review. This derivation should be useful in formulating novel computational methods for predicting binding affinities. It also permits several important points to be established. For example, it is found that the double-annihilation method of computing binding energy does not yield the standard free energy of binding, but can be modified to yield this quantity. The derivation also makes it possible to define clearly the changes in translational, rotational, configurational, and solvent entropy upon binding. It is argued that molecular mass has a negligible effect upon the standard free energy of binding for biomolecular systems, and that the cratic entropy defined by Gurney is not a useful concept. In addition, the use of continuum models of the solvent in binding calculations is reviewed, and a formalism is presented for incorporating a limited number of solvent molecules explicitly.

Binding Sites↗
PubMed1997

A new class of models for computing receptor-ligand binding affinities.

Models for predicting the binding affinities of molecules in solution are either very detailed, making them computationally intensive and hard to test, or very simple, and thus less informative than one might wish. A new class of models that focus on the predominant states of the binding molecules promise to capture the essential physics of binding at modest computational cost.

Kinetics↗
PubMed1996

The determinants of pKas in proteins.

Although validation studies show that theoretical models for predicting the pKas of ionizable groups in proteins are increasingly accurate, a number of important questions remain: (1) What factors limit the accuracy of current models? (2) How can conformational flexibility of proteins best be accounted for? (3) Will use of solution structures in the calculations, rather than crystal structures, improve the accuracy of the computed pKas? and (4) Why does accurate prediction of protein pKas seem to require that a high dielectric constant be assigned to the protein interior? This paper addresses these and related issues. Among the conclusions are the following: (1) computed pKas averaged over NMR structure sets are more accurate than those based upon single crystal structures; (2) use of atomic parameters optimized to reproduce hydration energies of small molecules improves agreement with experiment when a low protein dielectric constant is assumed; (3) despite use of NMR structures and optimized atomic parameters, pKas computed with a protein dielectric constant of 20 are more accurate than those computed with a low protein dielectric constant; (4) the pKa shifts in ribonuclease A that result from phosphate binding are reproduced reasonably well by calculations; (5) the substantial pKa shifts observed in turkey ovomucoid third domain result largely from interactions among ionized groups; and (6) both experimental data and calculations indicate that proteins tend to lower the pKas of Asp side chains but have little overall effect upon the pKas of other ionizable groups.

Amino Acid Sequence↗
PubMed1996

Binding of tacrine and 6-chlorotacrine by acetylcholinesterase.

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 deltaG 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 pronated inhibitor molecule and adjacent aromatic groups of Phe-330 and Trp-84.

Acetylcholinesterase↗
PubMed1995

Simulation of charge-mutant acetylcholinesterases.

A recent experimental study of human acetylcholinesterase has shown that the mutation of surface acidic residues has little effect on the rate constant for hydrolysis of acetylthiocholine. It was concluded, on this basis, that the reaction is not diffusion controlled and that electrostatic steering plays only a minor role in determining the rate. Here we examine this issue through Brownian dynamics simulations on Torpedo californica acetylcholinesterase in which the surface acidic residues homologous with those mutated in the human enzyme are artificially neutralized. The computed effects of the mutations on the rate constants reproduce quite well the modest effects of the mutations upon the measured encounter rates. Nonetheless, the electrostatic field of the enzyme is found to increase the rate constants by about an order of magnitude in both the wild type and the mutants. We therefore conclude that the mutation experiments do not disprove that electrostatic steering substantially affects the catalytic rate of acetylcholinesterase.

Acetylcholinesterase↗
PubMed1995

Theory of electrostatic interactions in macromolecules.

In the past year, substantial progress has been made in the modeling of electrostatic interactions in biomolecules. This review highlights advances in the following areas: first, the efficient computation of long-range electrostatic interactions in detailed molecular simulations; second, the application of the Poisson-Boltzmann electrostatic model in conformational analysis; third, the application of the Poisson-Boltzmann model in quantum chemistry calculations; fourth, the development of atomic parameters; and finally, the modeling of ionization equilibria in proteins.

Computer Simulation↗
PubMed1995

Acetylcholinesterase: diffusional encounter rate constants for dumbbell models of ligand.

For some enzymes, virtually every substrate molecule that encounters the entrance to the active site proceeds to reaction, at low substrate concentrations. Such diffusion-limited enzymes display high apparent bimolecular rate constants ((kcat/KM)), which depend strongly upon solvent viscosity. Some experimental studies provide evidence that acetylcholinesterase falls into this category. Interestingly, the asymmetric charge distribution of acetylcholinesterase, apparent from the crystallographic structure, suggests that its electrostatic field accelerates the encounter of its cationic substrate, acetylcholine, with the entrance to the active site. Here we report simulations of the diffusion of substrate in the electrostatic field of acetylcholinesterase. We find that the field indeed guides the substrate to the mouth of the active site. The computed encounter rate constants depend upon the particular relative geometries of substrate and enzyme that are considered to represent successful encounters. With loose reaction criteria, the computed rates exceed those measured experimentally, but the rate constants vary appropriately with ionic strength. Although more restrictive reaction criteria lower the computed rates, they also lead to unrealistic variation of the rate constants with ionic strength. That these simulations do not agree well with experiment suggests that the simple diffusion model is incomplete. Structural fluctuations in the enzyme or events after the encounter may well contribute to rate limitation.

Acetylcholine↗
PubMed1994

Prediction of pH-dependent properties of proteins.

We describe what may be the most accurate approach currently available for the calculation of the pKas of ionizable groups in proteins. The accuracy is assessed by comparison of computed pKas with 60 measured pKas in a total of seven proteins. The overall root-mean-square error is 0.89 pKa units. Linear regression analysis of computed versus measured pKas yields a slope of 0.95, y-intercept of -0.02 and a correlation coefficient of 0.96. The proposed approach also picks out many of the shifted pKas of groups in enzyme active sites and special salt bridges. However, it does yield several over-shifted pKas and tends to underestimate pKa shifts which result from desolvation effects. We examine the ability of the new approach to reproduce the dependence of protein stability upon pH, using the ionization polynomial formalism. Overall features of the stability curves are reproduced, but the quantitative agreement is not particularly good. The reasons for the disagreement may have to do both with insufficient accuracy in the theory and with uncertainty in the nature of the unfolded state of proteins. The methodology described here is based upon finite difference solutions of the Poisson-Boltzmann equation. Its success depends upon the use of the rather high protein dielectric constant of 20. However, theoretical considerations and the fact that pKa shifts which result from desolvation are underestimated here imply that the dielectric constant of the protein interior actually is lower than 20. We suggest that the high protein dielectric constant improves the overall agreement with experiment because it accounts approximately for phenomena which tend to mitigate pKa shifts and which are not specifically included in the model. These include conformational relaxation and specific ion-binding. Future models based upon a low protein dielectric constant and treating such phenomena explicitly might yield improved agreement with experiment.

Amino Acids↗
PubMed1994

Open "back door" in a molecular dynamics simulation of acetylcholinesterase.

The enzyme acetylcholinesterase generates a strong electrostatic field that can attract the cationic substrate acetylcholine to the active site. However, the long and narrow active site gorge seems inconsistent with the enzyme's high catalytic rate. A molecular dynamics simulation of acetylcholinesterase in water reveals the transient opening of a short channel, large enough to pass a water molecule, through a thin wall of the active site near tryptophan-84. This simulation suggests that substrate, products, or solvent could move through this "back door," in addition to the entrance revealed by the crystallographic structure. Electrostatic calculations show a strong field at the back door, oriented to attract the substrate and the reaction product choline and to repel the other reaction product, acetate. Analysis of the open back door conformation suggests a mutation that could seal the back door and thus test the hypothesis that thermal motion of this enzyme may open multiple routes of access to its active site.

Acetylcholine↗