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

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.