Coupling between local structure and global stability of a protein: mutants of staphylococcal nuclease

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Abstract
Staphylococcal nuclease exists in solution as a mixture of two folded (N and N'') and two unfolded (U and U*) forms. Earlier workers [Evans et al. (1989) Biochemistry 28, 362] have proposed that the N''/N and U/U* structural differences involve cis/trans isomerization about the Lys116-Pro117 peptide bond with N and U cis and N'' and U* trans. The present results show that residue changes throughout the nuclease structure have large effects on the distribution of the N and N'' forms. The N''/N ratios at 313 K for nuclease H124L (N''/N = 0.07) and nuclease G79S (N''/N = 12) differ by 2 orders of magnitude. Thermodynamic parameters for equilibria linking the two folded and two unfolded substates were evaluated for seven mutants of nuclease which were found by kinetic assays to have similar enzymatic activities but by NMR spectroscopy to have a wide dispersion of thermal stabilities. Our results indicate that mutational perturbations of the N''/N equilibrium in folded nuclease (.DELTA.G for the N .dblarw. N'' reaction) are strongly coupled to changes in the stability of the N form (.DELTA.G for the N .dblarw. U reaction), but much less so to the stability of the N'' form (.DELTA.G for the N'' .dblarw. U* reaction).