STABILIZATION OF MYOGLOBIN BY MULTIPLE ALANINE SUBSTITUTIONS IN HELICAL POSITIONS

We have carried out a series of multiple Xaa --> Ala changes at nonadj acent surface positions in the sequence of sperm whale myoglobin. Alth ough the corresponding single substitutions do not increase the therma l stability of the protein, multiple substitutions enhance the stabili ty of the resulting myoglobins. The effect observed is an increase in the observed T-m (midpoint unfolding temperature) relative to that pre dicted from assuming additivity of the free energy changes correspondi ng to single mutations. The stabilization occurs in the presence of ur ea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different he lices and are not close in sequence or in the native structure of myog lobin. The observed effect is consistent with a role of multiple alani nes in residual interactions in the unfolded state of the mutant prote ins.