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Surface point mutations that significantly alter the structure and stability of a protein's denatured state
Author(s) -
Smith Catherine K.,
Bu Zimei,
Engelman Donald M.,
Regan Lynne,
Anderson Karen S.,
Sturtevant Julian M.
Publication year - 1996
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560051007
Subject(s) - guanidine , circular dichroism , mutant , denaturation (fissile materials) , chemistry , radius of gyration , crystallography , protein folding , equilibrium unfolding , differential scanning calorimetry , native state , molten globule , point mutation , protein secondary structure , protein tertiary structure , biophysics , biochemistry , biology , organic chemistry , polymer , nuclear chemistry , physics , thermodynamics , gene
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.