To quantitate the contributions of the large hydrophobic residues in staphylococcal nuclease to the stability of its native state, single alanine and glycine substitutions were constructed by site-directed mutagenesis for each of the 11 leucine, 9 valine, 7 tyrosine, 5 isoleucine, 4 methionine, and 3 phenylalanine residues. In addition, each isoleucine was also mutated to valine. The resulting collection of 83 mutant nucleases was submitted to guanidine hydrochloride denaturation using intrinsic tryptophan fluorescence to monitor the equilibrium constant between the native and denatured states. From analysis of these data, each mutant protein’s stability to reversible denaturation [formula omitted] and sensitivity to guanidine hydrochloride [formula omitted] or d(ΔG)/d [GuHCl]) were obtained. Four unexpected trends were observed. (1) A striking bipartite distribution was found for sites of mutations that altered [formula omitted]: mutations that increased this parameter only involved residues that contribute side chains to the major hydrophobic core centered around a five-strand β-barrel, whereas mutations that caused β to decrease clustered around a second, smaller and less well-defined hydrophobic core. (2) The average stability loss for mutants in each of the six residue classes was 2–3 times greater than that estimated on the basis of the free energy of transfer of the hydrophobic side chain from water to n-octanol. (3) The magnitude of the stability loss on substituting Ala or Gly for a particular type of amino acid varied extensively among the different sites of its occurrence in nuclease, indicating that the environment surrounding a specific residue determines how large a stability contribution its side chain will make. On the basis of statistical analyses, the parameter that provided the best estimate of this environmental effect on ΔΔG is the number of Cα carbons within a sphere of 10-Å radius. (4) A significant correlation was found between the absolute value of the change in mGuHCl and the loss of stability. This correlation strongly supports the conclusion that amino acid substitutions can destabilize a protein indirectly via their effects on the structure and free energy of the denatured state.
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