Redox active transition metals such as copper present a dilemma to cell: they are dangerous but useful cofactors. Recent discoveries indicate that the cytoplasm of eukaryotic cells has an overcapacity for copper chelation, leaving open the question of how copper proteins obtain this essential cofactor. The long term goal of these studies is to elucidate the intacellular chemistry and cell biology of copper in yeast and humans. Our approach focuses on small soluble metal receptors known as metallochaperones. The metallochaperone hypothesis, described first for the Atx l protein, invokes a class of cytoplasmic metal receptor proteins that function in a 'chaperone-like' manner to guide and protect the metal ion while facilitating appropriate partnerships. The metallochaperone proteins characterized to date, Atx1 and CCS, clearly do not function to protect the cell from metal or oxygen toxicity. Instead, these proteins appear to ensure the safe delivery of the metal ion to its proper intracellular destination. In the process, they this protect the cargo from adventitious reactions and a multitude of alternative binding sites. Elucidating the mechanisms of this processes will provide keys to understanding the chemistry and biology of copper in pathological conditions that involve disorders of metal metabolism such as Wilson and Menkes disease and possibly some forms of amyotrophic lateral sclerosis (ALS). The proposed research test a variety structure-function aspects of human Atx1 and CCS and their interaction with known physiological targets including the Wilson and Menkes disease proteins. Energetic and structural aspects of the chaperone partnerships and metal transfer reactions will be resolved. Both thermodynamic and kinetic mechanism experiments have been designed to test the hypothesis that these metallochaperones function like enzymes: they lower the activation barrier for copper transfer along specific reaction coordinates (i.e., transfer to partner) but maintain higher barriers for transfer to non partner sites. If the proposal that the cytoplasm has an extraordinary overcapacity for copper chelation, then the dynamic aspects of metallochaperone structure and metal transfer chemistry will play a central role in copper trafficking within the cell.
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