Abstract:
:In order to quantitate the contributions of the polar, uncharged amino acids to the stability of the native state of staphylococcal nuclease, each of the 13 alanines, 9 glycines, 9 threonines, 6 prolines, 6 glutamines, 6 asparagines, and 3 serines was substituted, either with both alanine and glycine or with 1 of these 2 amino acids plus valine. For each mutant, the stability to reversible denaturation (delta GH2O) was quantitated by determining the Kapp for this reaction as a function of guanidine hydrochloride concentration. In addition, the parameter mGuHCl (= d(delta G)/d[GuHCl]) was calculated from the data. To identify the local structural features responsible for the relatively large and variable changes in delta GH2O and mGuHCl observed for the same type of substitution at different locations in nuclease, statistical correlations were sought between delta GH2O, mGuHCl, and a number of descriptors of the local structure. As with substitutions of the large hydrophobic amino acids [Shortle, D., Stites, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041], mutation of polar, uncharged residues to Gly leads to a change in stability that, on average, correlates well with the degree to which the wild-type residue is buried. This correlation is especially significant for threonine, an amino acid with both polar and hydrophobic character, but is not demonstrated for the more typically hydrophobic residue alanine. As reported in the previous study of alanine/glycine substitutions of hydrophobic residues, a significant correlation between changes in stability and changes in the value of mGuHCl is again observed, strengthening the conclusion that the putative structural changes in the denatured state which lead to increases or decreases in mGuHCl are responsible for a significant fraction of the stability loss for an average mutant. The existence of this correlation is consistent with the denatured state of wild-type staphylococcal nuclease having evolved to a relatively high free energy via optimization of a balance between a maximal exposure of hydrophobic surface and a minimal gain in chain entropy. On average, mutations are less stable in proportion to the extent of which they perturb this balance. A new and puzzling correlation is reported between the extent of buriedness of a residue in the wild-type native state versus the difference in mGuHCl between the Ala mutation and the Gly mutation at that position.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Green SM,Meeker AK,Shortle Ddoi
10.1021/bi00140a005subject
Has Abstractpub_date
1992-06-30 00:00:00pages
5717-28issue
25eissn
0006-2960issn
1520-4995journal_volume
31pub_type
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