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ABSTRACT: The binding thermodynamics of the HIV-1 protease inhibitor acetyl pepstatin and the substrate Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln, corresponding to one of the cleavage sites in the gag, gag-pol polyproteins, have been measured by direct microcalorimetric analysis. The results indicate that the binding of the peptide substrate or peptide inhibitor is entropically driven; i.e., it is characterized by an unfavorable enthalpy and a favorable entropy change, in agreement with a structure-based thermodynamic analysis based upon an empirical parameterization of the energetics. Dissection of the binding enthalpy indicates that the intrinsic interactions are favorable and that the unfavorable enthalpy originates from the energy cost of rearranging the flap region in the protease molecule. In addition, the binding is coupled to a negative heat capacity change. The dominant binding force is the increase in solvent entropy that accompanies the burial of a significant hydrophobic surface. Comparison of the binding energetics obtained for the substrate with that obtained for synthetic nonpeptide inhibitors indicates that the major difference is in the magnitude of the conformational entropy change. In solution, the peptide substrate has a higher flexibility than the synthetic inhibitors and therefore suffers a higher conformational entropy loss upon binding. This higher entropy loss accounts for the lower binding affinity of the substrate. On the other hand, due to its higher flexibility, the peptide substrate is more amenable to adapt to backbone rearrangements or subtle conformational changes induced by mutations in the protease. The synthetic inhibitors are less flexible, and their capacity to adapt is more restricted. The expected result is a more pronounced effect of mutations on the binding affinity of the synthetic inhibitors. On the basis of the thermodynamic differences in the mode of binding of substrate and synthetic inhibitors, it appears that a key factor to understanding resistance is given by the relative balance of the different forces that contribute to the binding free energy and, in particular, the balance between conformational and solvation entropy.
Biochemistry 05/1998; 37(17):5791-7. · 3.42 Impact Factor
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Advances in experimental medicine and biology 02/1998; 436:325-8. · 1.09 Impact Factor
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ABSTRACT: The prediction of binding affinities from structure is a necessary requirement in the development of structure-based molecular design strategies. In this paper, a structural parameterization of the energetics previously developed in this laboratory has been incorporated into a molecular design algorithm aimed at identifying peptide conformations that minimize the Gibbs energy. This approach has been employed in the design of mutants of the aspartic protease inhibitor pepstatin A. The simplest design strategy involves mutation and/or chain length modification of the wild-type peptide inhibitor. The structural parameterization allows evaluation of the contribution of different amino acids to the Gibbs energy in the wild-type structure, and therefore the identification of potential targets for mutation in the original peptide. The structure of the wild-type complex is used as a template to generate families of conformational structures in which specific residues have been mutated. The most probable conformations of the mutated peptides are identified by systematically rotating around the side-chain and backbone torsional angles and calculating the Gibbs potential function of each conformation according to the structural parametrization. The accuracy of this approach has been tested by chemically synthesizing two different mutants of pepstatin A. In one mutant, the alanine at position five has been replaced by a phenylalanine, and in the second one a glutamate has been added at the carboxy terminus of pepstatin A. The thermodynamics of association of pepstatin A and the two mutants have been measured experimentally and the results compared with the predictions. The difference between experimental and predicted Gibbs energies for pepstatin A and the two mutants is 0.23 +/- 0.06 kcal/mol. The excellent agreement between experimental and predicted values demonstrates that this approach can be used in the optimization of peptide ligands.
Proteins Structure Function and Bioinformatics 02/1998; 30(1):74-85. · 3.39 Impact Factor
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ABSTRACT: Two effects are mainly responsible for the observed enthalpy change in protein unfolding: the disruption of internal interactions within the protein molecule (van der Waals, hydrogen bonds, etc.) and the hydration of the groups that are buried in the native state and become exposed to the solvent on unfolding. In the traditional thermodynamic analysis, the effects of hydration have usually been evaluated using the thermodynamic data for the transfer of small model compounds from the gas phase to water. The contribution of internal interactions, on the other hand, are usually estimated by subtracting the hydration effects from the experimental enthalpy of unfolding. The main drawback of this approach is that the enthalpic contributions of hydration, and those due to the disruption of internal interactions, are more than one order of magnitude larger than the experimental enthalpy value. The enthalpy contributions of hydration and disruption of internal interactions have opposite signs and cancel each other almost completely resulting in a final value that is over 10 times smaller than the individual terms. For this reason, the classical approach cannot be used to accurately predict unfolding enthalpies from structure: any error in the estimation of the hydration enthalpy will be amplified by a factor of 10 or more in the estimation of the unfolding enthalpy. Recently, it has been shown that simple parametric equations that relate the enthalpy change with certain structural parameters, especially changes in solvent accessible surface areas have considerable predictive power. In this paper, we provide a physical foundation to that parametrization and in the process we present a system of equations that explicitly includes the enthalpic effects of the packing density between the different atoms within the protein molecule. Using this approach, the error in the prediction of folding/unfolding enthalpies at 60 degrees C, the median temperature for thermal unfolding, is better than +/- 3% (standard deviation = 4 kcal/mol).
Proteins Structure Function and Bioinformatics 11/1996; 26(2):123-33. · 3.39 Impact Factor
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ABSTRACT: The magnitude of the conformational entropy change experienced by the peptide backbone upon protein folding was investigated experimentally and by computational analysis. Experimentally, two different pairs of mutants of a 33 amino acid peptide corresponding to the leucine zipper region of GCN4 were used for high-sensitivity microcalorimetric analysis. Each pair of mutants differed only by having alanine or glycine at a specific solvent-exposed position under conditions in which the differences in stability could be attributed to differences in the conformational entropy of the unfolded state. The mutants studied were characterized by different stabilities but had identical heat capacity changes of unfolding (delta Cp), identical solvent-related entropies of unfolding (delta Ssolv), and identical enthalpies of unfolding (delta H) at equivalent temperatures. Accordingly, the differences in stability between the different mutants could be attributed to differences in conformational entropy. The computational studies were aimed at generating the energy profile of backbone conformations as a function of the main chain dihedral angles phi and phi. The energy profiles permit a direct calculation of the probability distribution of different conformers and therefore of the conformational entropy of the backbone. The experimental results presented in this paper indicate that the presence of the methyl group in alanine reduces the conformational entropy of the peptide backbone by 2.46 +/- 0.2 cal/K. mol with respect to that of glycine, consistent with a 3.4-fold reduction in the number of allowed conformations in the alanine-containing peptides. Similar results were obtained from the energy profiles. The computational analysis also indicates that the addition of further carbon atoms to the side chain had only a small effect as long as the side chains were unbranched at position beta. A further reduction with respect to Ala of only 0.61 and 0.81 cal/K. mol in the backbone entropy was obtained for leucine and lysine, respectively. beta-branching (Val) produces the largest decrease in conformational entropy (1.92 cal/K.mol less than Ala). Finally, the backbone entropy change associated with the unfolding of an alpha-helix is 6.51 cal/K.mol for glycine. These and previous results have allowed a complete estimation of the conformational entropy changes associated with protein folding.
Proteins Structure Function and Bioinformatics 07/1996; 25(2):143-56. · 3.39 Impact Factor
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ABSTRACT: The discovery that the protease from the human immunodeficiency virus (HIV) belongs to the aspartic protease family has generated renewed interest in this class of proteins. In this paper, the interactions of endothiapepsin, an aspartic proteinase from the fungus Endothia parasitica, with the inhibitor pepstatin A have been studied by high-sensitivity calorimetric techniques. These experiments have permitted a complete characterization of the temperature and pH-dependence of the binding energetics. The binding reaction is characterized by negative intrinsic binding enthalpy and negative heat capacity changes. The association constant is maximal at low pH (2 x 10(9) M-1 at pH 3) but decreases upon increasing pH (8.1 x 10(6) M-1 at pH 7). The binding of the inhibitor is coupled to the protonation of one of the aspartic moieties in the Asp dyad of the catalytic site of the protein. This phenomenon is responsible for the decrease in the apparent affinity of the inhibitor for the enzyme upon increasing pH. The experimental results presented here indicate that the binding of the inhibitor is favored both enthalpically and entropically. While the favorable enthalpic contribution is intuitively expected, the favorable entropic contribution is due to the large gain in solvent-related entropy associated with the burial of a large hydrophobic surface, that overcompensates the loss in conformational and translational/rotational degrees of freedom upon complex formation. The characteristics of the molecular recognition process have been evaluated by means of structure-based thermodynamic analysis. Three regions in the protein contribute significantly to the free energy of binding: the residues surrounding the Asp dyad (Asp32 in the N-terminal lobe and Asp215 in the C-terminal domain) and the flap region (Ile73 to Asp77). In addition, the rearrangement of residues that are not in immediate contact with the inhibitor provides close to 40% of the protease contribution to the binding free energy. On the other hand, the two statine residues provide more than half of the inhibitor contributions to the total free energy of binding. It is demonstrated that a previously developed empirical structural parametrization of the thermodynamic parameters that define the Gibbs energy, accurately accounts for the binding energetics and its temperature and pH-dependence.
Journal of Molecular Biology 10/1995; 252(3):337-50. · 4.00 Impact Factor
