Guanidine hydrochloride denaturation studies of mutant forms of staphylococcal nuclease

Publisher Website
Google Scholar
Abstract
Several mutant forms of Staphylococcal nuclease with one or two defined amino acid substitutions have been purified, and the effects of the altered amino acid sequence on the stability of the folded conformation have been analyzed by guanidine hydrochloride denaturation. Two nuc‐ mutations, which greatly reduced the level of enzyme activity accumulated in Ecoli colonies carrying a recombinant plasmid with the mutant nuc gene (i.e., a NUC‐phenotype), both result in protein unfolding at significantly lower guanidine hydrochloride concentrations than the wild‐type protein, whereas three sup mutations isolated on the basis of their ability to suppress partially the NUC‐phenotype of the above two mutations result in unfolding at significantly higher guanidine hydrochloride concentrations. Characterization of nuclease molecules with two different amino acid substitutions, either nuc− + sup pairs or sup + sup pairs, suggests that the effect of an amino acid substitution on the stability of the native conformation, as measured by the value of ΔΔGD, may not be a constant, but rather a variable that is sensitive to the presence of other substitutions at distant sites in the same molecule. Surprisingly, the slopes of the log Kapp vs guanidine hydrochloride concentration plots vary by as much as 35% among the different proteins.