PROTEIN STABILITY FOR SINGLE SUBSTITUTION MUTANTS AND THE EXTENT OF LOCAL COMPACTNESS IN THE DENATURED STATE

The stability changes caused by single amino acid substitutions are st udied by a simple, empirical method which takes account of the free en ergy change in the compact denatured state as well as in the native st ate. The conformational free energy is estimated from effective inter- residue contact energies, as evaluated in our previous study. When thi s method is applied, with a simple assumption about the compactness of the denatured state, for single amino acid replacements at Glu49 of t he tryptophan synthase a subunit and at Ile3 of bacteriophage T4 lysoz yme, the estimates of the unfolding Gibbs free energy changes correlat e well with observed values, especially for hydrophobic amino acids, a nd it also yields the same magnitudes of energy as the observed values for both proteins. When it is also applied for amino acid replacement s at various positions to estimate the average number of contacts at e ach position in the denatured state from the observed value of unfoldi ng free energy change, those values for replacements with Gly and Ala at the same residue position in staphylococcal nuclease correlate well with each other. The estimated numbers of contacts indicate that the protein is not fully expanded in the denatured state and also that the compact denatured state may have a substantially native-like topology , like the molten globule state, in that there is a weak correlation b etween the estimated average number of contacts at each residue positi on in the denatured state and the number of contacts in the native str ucture. These results provide some further evidence that the inter-res idue contact energies as applied here (i) properly reflect actual inte r-residue interactions and (ii) can be considered to be a pairwise hyd rophobicity scale. Also, the results indicate that characterization of the denatured state is critical to understanding the folding process.