Effects of chemical modifications of staphylococcal nuclease.

摘要

This thesis describes work on the stabilities and structural effects on staphylococcal nuclease by chemical modification of cysteine and/or methionine residues. The well characterized model protein, staphylococcal nuclease was used to explore the effect upon protein structural stability of oxidizing methionine and cysteine. These effects were compared to the effects of substituting methionines with isoleucine and leucine, a potential strategy for stabilizing proteins against oxidative damage. Stabilities of both oxidized and unoxidized proteins were determined by guanidine hydrochloride denaturation. Comparison of mutant protein's stability losses upon oxidation showed that methionine 26 or 32 had little destabilizing effect when oxidized. 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.; The oxidation of buried cysteine or methionine residues obviously can disrupt structure. Engineering in such an oxidatively triggered switch would only be useful if the effects of substitution are relatively minor, while the effects of the oxidized side chain upon structure are significant and the oxidation relatively easy. To assess the feasibility of this strategy, the effects of such substitutions and their oxidation were studied. Cysteines were found to be generally well tolerated in buried positions but these mutants were not more destabilized than wild-type when oxidized. Since the effect on structural stability of oxidizing a buried cysteine is expected to be large, this implies that buried cysteines are difficult to oxidize. Similar effects were observed for methionine.; A series of crosslinkers of various length were synthesized and used to make covalently linked dimers of our model protein, staphylococcal nuclease, in order to explore the effects of altering the denatured state of this protein. The guanidine hydrochloride denaturation data from the crosslinked proteins fits a three-state model. The thermal denaturation data fits a two-state model, but with obvious correlations between the thermal and solvent denaturation data. These thermodynamic changes appear to be due to excluded volume effects in the denatured state.