Abstract：
:The importance of two buried salt bridges in barnase in the stability of its folded state, the major transition rate for unfolding, and a folding intermediate has been analyzed by protein engineering, kinetic, and thermodynamic studies. The aspartate residues in the bridges Arg69-Asp93 and Arg83-Asp75 were replaced by the isosteric analogue asparagine, while various replacements were probed for the positively charged arginine partners. The mutations are very destabilizing, lowering stability by up to 5.4 kcal/mol. A value of 3.0-3.5 kcal/mol was derived for the coupling energy between Arg and Asp from a double mutant cycle analysis. Despite the radical nature of these mutations, they do not appear to alter the pathway of folding. The interaction between Arg69 and Asp93, located in a relatively conserved region among ribonucleases, is predominantly formed in the major transition state along the folding pathway, as found previously from an analysis of more benign mutations; the value of phi(F) for all mutations at positions 69 and 93 are 0.8-0.9 in the major transition state for folding where phi(F) = 0 = fully unfolded and phi(F) = 1 = fully folded interaction energies). In contrast, the interaction between Arg83 and Asp75 in the active site of barnase is formed only in the native state of the protein. The analysis of folding pathways and the structure of folding intermediates by making kinetic and thermodynamic measurements on mutants appears even more robust than expected.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Tissot AC,Vuilleumier S,Fersht ARdoi
10.1021/bi952930esubject
Has Abstractpub_date
1996-05-28 00:00:00pages
6786-94issue
21eissn
0006-2960issn
1520-4995pii
bi952930ejournal_volume
35pub_type
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