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 sensitive to oxidat ive damage caused by a variety of oxidants, The model protein staphylococca l nuclease was used to explore the effect on protein structural stability o f oxidizing methionine to the sulfoxide form. These effects were compared w ith the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Wild- type nuclease and various mutants were oxidized with hydrogen peroxide, Sta bilities of both oxidized and unoxidized proteins were determined by guanid ine hydrochloride denaturation, Oxidation destabilized the wild-type protei n by over 4 kcal/ mel. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the pr otein native state. Comparison of mutant protein's stability losses upon ox idation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32, While substitution of methi onine 98 carried as great an energetic penalty as oxidation, substitution a t position 65 was less disruptive than oxidation, Thus a simple substitutio n mutagenesis strategy to protect a protein against oxidative destabilizati on is practical for some methionine residues.