IMPORTANCE OF 2 BURIED SALT BRIDGES IN THE STABILITY AND FOLDING PATHWAY OF BARNASE

The importance of two buried salt bridges in barnase in the stability of its folded state, the major transition state for unfolding, and a f olding intermediate has been analyzed by protein engineering, kinetic, and thermodynamic studies. The aspartate residues in the bridges Arg6 9-Asp93 and Arg83-Asp75 were replaced by the isosteric analogue aspara gine, while various replacements were probed for the positively charge d arginine partners. The mutations are very destabilizing, lowering st ability 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 cycl e 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 ribonucleas es, is predominantly formed in the major transition state along the fo lding pathway, as found previously from an analysis of more benign mut ations; the value of Phi(F) for all mutations at positions 69 and 93 a re 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). I n contrast, the interaction between Arg83 and Asp75 in the active site of barnase is formed only in the native state of the protein. The ana lysis of folding pathways and the structure of folding intermediates b y making kinetic and thermodynamic measurements on mutants appears eve n more robust than expected.