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Importance of Two Buried Salt Bridges in the Stability and Folding Pathway of Barnase

1996; American Chemical Society; Volume: 35; Issue: 21 Linguagem: Inglês

10.1021/bi952930e

1943-295X

A. C. Tissot, Stéphane Vuilleumier, A. Fersht,

Hemoglobin structure and function

The importance of two buried salt bridges in barnase in the stability of its folded state, the major transition state 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 ΦF for all mutations at positions 69 and 93 are 0.8−0.9 in the major transition state for folding (where ΦF = 0 = fully unfolded and ΦF = 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.