Oakwood College1, Department of Biological Sciences, Huntsville, AL 35896 Western Michigan University2, Department of Chemistry, Kalamazoo, MI 49008
Wilson’s disease is an autosomoal recessive disorder that is mapped to gene ATP7B on chromosome 13, it is also one of the most prevalent copper metabolism disorders. The Wilson’s protein is predominately responsible for the copper shuttling and excretion when in excess. Consisting of four major domains, our interests lie in the N-terminal copper-binding domain containing six separate copper binding domains. Once inside the cell, copper is acquired by different copper specific proteins that shuttle it to predetermined destinations; hCTR1 functions to transport copper into the cell, CCS serves to shuttle copper to superoxide dismutase, while HAH1 transports copper to the Wilson’s protein. Copper transfer studies show that HAH1, a copper metallochaperone, has the ability to transfer copper to any one of the first four domains on the N-terminus, Wilson’s domain 1-4 (WD#), with WD4 displaying the highest affinity for Cu(I)-HAH1. Studies suggest that copper transfer from HAH1 to WD4 occurs through a 3-coordinate copper mediated protein-protein interaction. It is our goal to characterize this proposed protein-protein complex between Cu(I)-HAH1 and WD4 employing both native and mutated forms of HAH1 and WD4. To produce the proteins of interest, gene constructs of both HAH1-C11A and WD4 underwent transformation and protein expression. The proteins were then purified and concentration was determined through Bradford Assay and Thiol quantitation. The proteins are now available for titration studies to capture HAH1-WD4 interaction. Possible adducts will be analyzed using UV, XANES/EXAFS, and NMR.
L. Walwyn-Tross was supported by NSF-DBI 0552517