Selected mutations in a mesophilic cytochrome c confer the stability of a thermophilic counterpart

Mesophilic cytochrome c(551) of Pseudomonas aeruginosa (PA c(551)) became a s stable as its thermophilic counterpart, Hydrogenobacter thermophilus cyto chrome c(552) (HT c(552)), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selec ted and mutated with reference to the corresponding residues in HT c(552) t hrough careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c(551) could be partly attained through an enthalpic factor. The solution structure of the mutant showed t hat, as in HT c(552), there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provi de a novel example of protein stabilization in that limited amino acid subs titutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.