title: Comparing the effect on protein stability of methionine oxidation versus mutagenesis: steps toward engineering oxidative resistance in proteins.
abstract: The biological activity of some proteins is known to be sensitive to oxidative damage caused by a variety of oxidants. The model protein staphylococcal nuclease was used to explore the effect on protein structural stability of oxidizing methionine to the sulfoxide form. These effects were compared with 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. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Oxidation destabilized the wild-type protein by over 4 kcal/mol. This large loss of stability supports the idea that in some cases loss of biological activity is linked to disruption of the protein native state. Comparison of mutant protein's stability losses upon oxidation showed that methionines 65 and 98 had a much greater destabilizing effect when oxidized than methionines 26 or 32. While substitution of methionine 98 carried as great an energetic penalty as oxidation, substitution at position 65 was less disruptive than oxidation. Thus a simple substitution mutagenesis strategy to protect a protein against oxidative destabilization is practical for some methionine residues.
authors: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701-1021, USA.
authors: Y H
authors: YH
authors: Kim
authors: A H
authors: AH
authors: Berry
authors: D S
authors: DS
authors: Spencer
authors: W E
authors: WE
authors: Stites
mesh_terms: D004795:Enzyme Stability; D008715:Methionine / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering
chemical_list: D008715:Methionine; D008836:Micrococcal Nuclease