Surface point mutations that significantly alter the structure and stability of a protein's denatured state

Abstract

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 s-1 for the m+ mutant and 952 s-1 for the m- mutant) and the rates of unfolding (11 s-1 for m+ mutant and 3 s-1 for the m- mutant) were significantly different, as determined by stopped flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (α((+)) = 0.3). Small- angle X-ray scattering showed that the radius of gyration of the denatured state (R(gd)) of the m+ mutant did not change with increasing denaturant concentrations (R(gd) ≃ 23 Å); whereas, the R(gd) of the m- mutant increased from approximately 17 Å to 23 Å with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.