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 (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.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
C K
CK
Smith
Z
Z
Bu
K S
KS
Anderson
J M
JM
Sturtevant
D M
DM
Engelman
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L
Regan
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002151:Calorimetry; D002942:Circular Dichroism; D005470:Fluorometry; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D006146:Guanidines; C034284:IgG Fc-binding protein, Streptococcus; D019791:Guanidine