The contribution of the residues from the main hydrophobic core of ribonuclease A to its pressure-folding transition state.

The role of hydrophobic interactions established by the residues that belong to the main hydrophobic core of ribonuclease A in its pressure-folding transition state was investigated using the Phi-value method. The folding kinetics was studied using pressure-jump techniques both in the pressurization and depressurization directions. The ratio between the folding activation volume and the reaction volume (beta p-value), which is an index of the compactness or degree of solvation of the transition state, was calculated. All the positions analyzed presented fractional Phi f-values, and the lowest were those corresponding to the most critical positions for the ribonuclease A stability. The structure of the transition state of the hydrophobic core of ribonuclease A, from the point of view of formed interactions, is a relatively, uniformly expanded form of the folded structure with a mean Phi f-value of 0.43. This places it halfway between the folded and unfolded states. On the other hand, for the variants, the average of beta p-values is 0.4, suggesting a transition state that is 40% native-like. Altogether the results suggest that the pressure-folding transition state of ribonuclease A looks like a collapsed globule with some secondary structure and a weakened hydrophobic core. A good correlation was found between the Phi f-values and the Deltabeta p-values. Although the nature of the transition state inferred from pressure-induced folding studies and the results of the protein engineering method have been reported to be consistent for other proteins, to the best of our knowledge this is the first direct comparison using a set of mutants.