Attempts to Delineate the Relative Contributions of Changes in Hydrophobicity and Packing to Changes in Stability of Ribonuclease S Mutants

While the hydrophobic driving force is thought to be a majorcontributor to protein stability, it is difficult to experimentallydissect out its contribution to the overall free energy of folding. Wehave made large to small substitutions of buried hydrophobic residuesat positions 8 and 13 in the peptide/protein complex, RNase-S, and havecharacterized the structures by X-ray crystallography. Thethermodynamics of association of these mutant S peptides with S proteinwas measured in the presence of different concentrations of methanoland ethanol. The reduction in the strength of the hydrophobic drivingforce in the presence of these organic solvents was estimated fromsurface-tension data as well as from the dependence of the $\Delta C_p$ ofprotein/peptide binding on the alcohol concentration. The dataindicated a decrease in the strength of the hydrophobic driving forceof about 30-40% over a 0-30% range of the alcohol concentration. Weobserve that large to small substitutions destabilize the protein.However, the amount of destabilization, relative to the wild type, isindependent of the alcohol concentration over the range of alcoholconcentrations studied. The data clearly indicate that decreasedstability of the mutants is primarily due to the loss of packinginteractions rather than a reduced hydrophobic driving force andsuggest a value of the hydrophobic driving force of less than 18 cal mol^-^1 Å2