Biochemical Characterization of Domain Four of the Wilson Protein
Dickinson, Sarah E.
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The presence of copper in human cells is important for cell functions, such as mitochondrial respiration and amino acid metabolism. Wilson protein and the metallochaperone,HAHl, are responsible for the transportation of copper from the surface of the cell to its target inside the cell. HAHl transports copper from the cytosol to the copper-binding domains of Wilson protein. The interaction between HAHl and the copper-binding domains of Wilson protein involves the transfer of copper from HAHl to Wilson protein, which then transports the copper across the membranes of vesicles inside thecell. Mutationsin the gene that codes for Wilson protein inhibit the transportation of copper, resulting in Wilson Disease, which is characterized by the accumulation of copper in the brain and liver. The current study was undertaken to investigate the mechanism of the transition of copper from the surface of the cell to its target inside the cell, in particular,the transfer of the metal ion from the metallochaperone to the copper-binding domain four (WLN4). In order to characterize the protein complex intermediate, point mutations were introduced at the copper-binding sites of the chaperone that will prevent the complete transfer of copper from the chaperone to the protein and allow for the analysis of the chemistry involved in the transition. Two mutant proteins were successfully synthesized and purified for future analysis of the intermediate by NMR, XANES/EXAFS, and UV/VIS spectroscopy.