This proposal will investigate copper ion homeostasis and trafficking at the level of protein-protein interactions between the copper chaperone for Cu,Zn-superoxide dismutase (CCS) and Cu,Zn-superoxide dismutase (CuZnSOD). Copper is essential for biological function yet in its free state it is quite toxic to cells. It is this fundamental disparity that is the source of our investigations. How copper, a toxic metal ion in its free state, is distributed to distinct cellular locations and is used by particular proteins or in molecular mechanisms of protein-protein recognition and metal ion transfer without injury to the cell, is not well understood. In recent years an emerging family of copper chaperone proteins in eukayrotes has been identified which specifically aids in the regulation of copper ion transport. This proposal will investigate one of these copper chaperones, the copper chaperone for Cu,Zn-superoxide dismutase (CCS) which recognizes and specifically delivers copper ion to cytoplasmic Cu,Zn-superoxide dismutase (CuZnSOD). My experimental design will focus on, 1) expression, isolation, and structural determination of the full length and truncated forms of CCS from a variety of species, alone and in complex with their cognate CuZnSODs, 2) Elucidation of the solution properties of the CCS molecules and their complexes with CuZnSODs under different conditions, and 3) Determination of the stoichiometry of association of the CCS molecules and their complexes with CuZnSODs under different conditions in solution alone and in complex. This information should provide significant information about the molecular mechanisms used in the transfer of copper from CCS to SOD and provide a possible interesting target for novel therapeutic agents.
