Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins

The biological activity of some proteins is known to be sensitiveto oxidative damage caused by a variety of oxidants. The modelprotein staphylococcal nuclease was used to explore the effecton protein structural stability of oxidizing methionine to thesulfoxide form. These effects were compared with the effectsof substituting methionines with isoleucine and leucine, a potentialstrategy for stabilizing proteins against oxidative damage.Wild-type nuclease and various mutants were oxidized with hydrogenperoxide. Stabilities of both oxidized and unoxidized proteinswere determined by guanidine hydrochloride denaturation. Oxidationdestabilized the wild-type protein by over 4 kcal/mol. Thislarge loss of stability supports the idea that in some casesloss of biological activity is linked to disruption of the proteinnative state. Comparison of mutant protein's stability lossesupon oxidation showed that methionines 65 and 98 had a muchgreater destabilizing effect when oxidized than methionines26 or 32. While substitution of methionine 98 carried as greatan energetic penalty as oxidation, substitution at position65 was less disruptive than oxidation. Thus a simple substitutionmutagenesis strategy to protect a protein against oxidativedestabilization is practical for some methionine residues.