Contribution of the hydrophobic effect to protein stability: analysis based on simulations of the Ile-96----Ala mutation in barnase.

Abstract

Molecular dynamics simulations have been used to compute the difference in the unfolding free energy between wild-type barnase and the mutant in which Ile-96 is replaced by alanine. The simulations yield results (-3.42 and -5.21 kcal/mol) that compare favorably with experimental values (-3.3 and -4.0 kcal/mol). The major contributions to the free energy difference arise from bonding terms involving degrees of freedom of the mutated side chain and from nonbonded interactions of that side chain with its environment in the folded protein. By comparison with simulations of an extended peptide in the absence of solvent, used as a reference state, hydration effects are shown to play a minor role in the overall free energy balance for the Ile -* Ala transformation. The implications of these results for our understanding of the hydrophobic effect and its contribution to protein stability are discussed.Hydrophobic interactions are believed to make a major contribution to stabilizing the native structure of proteins in an aqueous environment (1-4). According to the classical picture (1) and more recent theoretical analyses (4), the hydrophobic effect leads to structures in which many, but not all, of the nonpolar side chains are packed together in the protein interior where they avoid contact with water. Measurements of solvation effects have shown that the hydrophobic effect is largely entropic in origin at room temperature (5). The unfavorable entropy of solvation is ascribed to the entropy decrease of water surrounding the nonpolar groups (6, 7); this is in accord with simulation studies of hydrophobic solutes (8, 9) and peptides in water (10).Although the general role of the hydrophobic effect in protein folding is understood, a more detailed quantitative description of its contributions to protein stability is essential. Site-directed mutagenesis experiments combined with thermal and spectroscopic stability measurements are now being used to dissect the contributions of individual amino acids (11). Substitutions of buried or partly buried nonpolar residues in several proteins (12-15) have shown that the change in thermodynamic stability between the wild type and mutant can be related to the free energies of transfer (16)(17)(18) and/or the accessible surface areas (19) of the individual substituted residues. Although there is a good overall correlation when a wide range of substitutions are considered (12), the variation seen among the hydrophobic aliphatic side chains does not show any simple relation with transfer free energies. Moreover, the interpretation is somewhat confused by the use of transfer free energy values from different solvents or from the gas phase to water for analyzing the experimental data (12,14). There are also more basic questions concerning the effect of hydrophobic solvation of individual nonpolar groups and its relationship to their bulk properties in the protein interior (20). Whether the protein interior corresponds to a nonpolar or a slightly polar liquid (e.g., alcohol-like) ...

Advances and Continuing Challenges in Achieving Realistic and Predictive Simulations of the Properties of Organic and Biological Molecules

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Restoring allosterism with compensatory mutations in hemoglobin.

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