Mutations in DJ-1, a protein involved in the oxidative stress response and mitochondrial function, cause a rare form of heritable parkinsonism. The oxidation of a conserved cysteine residue (C106) has been shown to be necessary for DJ-1-mediated cytoprotection, suggesting a mechanism by which DJ-1 senses the redox status of the cell. The pathogenic M26I mutation causes packing defects in the core of the protein that somehow disrupt the protein's protective function; however, M26I does not profoundly disrupt the homodimerization of DJ-1, and it is unknown why M26I DJ-1 is less stable in the cell. In order to probe the physical basis of DJ-1 dysfunction caused by the M26I mutation, we have incorporated a fluorescent reporter tryptophan (W) at four positions so that we may spectroscopically characterize the differences between the pathogenic M26I mutant and WT DJ-1 in their reduced and oxidized forms. In this study, we show that several of the W mutants have very modest effects on the thermal stability and secondary structure of DJ-1, indicating that they can serve as non-disruptive reporters of differences between WT and M26I DJ-1. Furthermore, results from fluorescence spectroscopy imply that there is a significant change in the environment of the L101 position between WT and M26I.

Introduction of tryptophan mutants into WT and M26I DJ-1 to probe differences in protein behavior

247th National Meeting (2014)

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