Contribution of Hydrophobic Residues to the Stability of Ribonuclease A Chain Folding Initiation Site Mutants. A Comparison of Pressure and Temperature Induced Unfolding

A set of ribonuclease A (RNase A) variants has been constructed within a postulated chain folding initiation site (CFIS) extending from residue 106 to 118 (of 124), in order to study the contributions of the hydrophobic residues to the stability of the protein. The free energy of unfolding of wild-type and mutants has been measured following the pressure and temperature induced equilibrium unfolding by fourth derivative absorption spectroscopy. The results obtained lay the foundations for future kinetic and structural studies of the CFIS and reinforce the potential of pressure as a denaturating agent for characterizing amino acid replacements.