ASP 79 makes a large, unfavorable contribution to the conformational stability of Ribonuclease SA

Abstract

An important unsolved problem in the field of biochemistry is the protein folding problem. Extensive research is being done to predict the structure and function of a protein given just its linear sequence of amino acids. The answer to this problem will lie in understanding the interactions involved in protein folding. Ribonuclease Sa (RNase Sa) is a relatively small protein with just 96 amino acids, and it is a good model for studying protein folding. The side-chain carboxyl of Asp 79 in RNase Sa is 89% buried, partially charged, and does not form any intramolecular hydrogen bonds. This amino acid was replaced by several amino acids including hydrophobic, polar uncharged, and positively charged amino acids, and the stability of the protein increased in each substitution that was introduced. The range of stability increase was from 1.7 to 2.9 kcal/mol. Thus, Asp 79 contributes unfavorably to the stability of the protein, and it is a good site for mutation in order to increase the stability of the protein. Also, the stability was measured as a function of pH for RNase Sa and the D79F mutant. The pK of Asp 79 was found to be 7.2 in the native state and 4.7 in the denatured state.