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 (alpha ++ = 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 approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A 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. Study holds ProTherm entries: 2983, 2984, 2985, 2986, 2987, 2988, 2989 Extra Details:

Submission Details

ID: kwSu3oDm

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:20 p.m.

Version: 1

Publication Details

Additional Information