NMR ANALYSIS OF THE RESIDUAL STRUCTURE IN THE DENATURED STATE OF AN UNUSUAL MUTANT OF STAPHYLOCOCCAL NUCLEASE

Background: Staphylococcal nuclease is a well-developed model system f or analyzing the effects of mutations on protein folding and stability . Substitution of glycine 88 with valine (Gly88Val) destabilizes staph ylococcal nuclease by 1.0 kcal mole(-1) and reduces its sensitivity to the denaturant guanidine hydrochloride, a phenomenon which may indica te an increase in residual structure in the denatured state. To assess its effects on denatured state structure, the Gly88Val mutation was i ncorporated into a 136 residue nonsense fragment which has been develo ped as a model of the wild type denatured state. Results: Application of two- and three-dimensional NMR spectroscopy to the Gly88Val fragmen t uniformly labeled with N-15 and C-13 has led to the assignment of 93 of the 136 residues. Comparison of chemical shifts of backbone resona nces to those of wild type native nuclease, analysis of the secondary shifts of the assigned resonances and nuclear Overhauser effects invol ving backbone protons indicate that, unlike the wild type fragment, mo st if not all of the five-stranded beta-barrel structure persists in t his denatured state. Conclusion: One major effect of the Gly88Val muta tion is to perturb the cooperative breakdown of the folded conformatio n, leading to a denatured state which is both more ordered and more st able than that formed by the wild type sequence. Since the equilibrium between the native and denatured states depends on the free energy di fference between them, stabilization of the denatured state by the Gly 88Val mutation indirectly destabilizes the native state.