AbstractSignificantly 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 (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd≈︁23 Å); whereas, the Rgd 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.

Smith, C. K., Bu, Z., Engelman, D. M., Regan, L., Anderson, K. S., & Sturtevant, J. M. (1996). Surface point mutations that significantly alter the structure and stability of a protein’s denatured state. Protein Science, 5(10), 2009–2019. Portico.