Protein stability for single substitution mutants and the extent of local compactness in the denatured state

The stability changes caused by single amino acid substitutionsare studied by a simple, empirical method which takes accountof the free energy change in the compact denatured state aswell as in the native state. The conformational free energyis estimated from effective inter-residue contact energies,as evaluated in our previous study. When this method is applied,with a simple assumption about the compactness of the denaturedstate, for single amino acid replacements at Glu49 of the tryptophansynthase subunit and at Ile3 of bacteriophage T4 lysozyme,the estimates of the unfolding Gibbs free energy changes correlatewell with observed values, especially for hydrophobic aminoacids, and it also yields the same magnitudes of energy as theobserved values for both proteins. When it is also applied foramino acid replacements at various positions to estimate theaverage number of contacts at each position in the denaturedstate from the observed value of unfolding free energy change,those values for replacements with Gly and Ala at the same residueposition in staphylococcal nuclease correlate well with eachother. The estimated numbers of contacts indicate that the proteinis not fully expanded in the denatured state and also that thecompact denatured state may have a substantially native-liketopology, like the molten globule state, in that there is aweak correlation between the estimated average number of contactsat each residue position in the denatured state and the numberof contacts in the native structure. These results provide somefurther evidence that the inter-residue contact energies asapplied here (i) properly reflect actual inter-residue interactionsand (ii) can be considered to be a pairwise hydrophobicity scale.Also, the results indicate that characterization of the denaturedstate is critical to understanding the folding process.