Copper containing proteins play important roles in organisms ranging from bacteria and yeast to plants and animals. Three different copper-containing proteins are the focus of this research project. The overall objectives are to understand the properties and biological functions of wild type copper-zinc superoxide dismutases (CuZn SOD), to understand why mutant human CuZn SOD proteins cause familial amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), to understand the properties and biological functions of stellacyanins and the copper binding domains of human blood coagulation factor VIII, and to prepare novel synthetic metalloproteins using the genes for CuZn SOD and stellacyanin as starting points. CuZnSOD is an antioxidant enzyme that catalyzes the disproportionation of superoxide. Approximately 50 different single mutations in human CuZn SOD have individually been linked to an inherited form of amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease). Results of transgenic mouse studies point to a toxic gain of function for these mutations rather than a loss of their antioxidant function as the cause of the disease. Human ALS mutant CuZn SODs have been prepared in our laboratory and have been found to have lost several of the properties characteristic of wild type CuZn SOD other than its SOD activity. These properties are highly likely to be essential to CuZnSOD in performing its normal biological functions. Moreover, the loss of these properties in the ALS mutant enzymes will likely prove to be linked to the gain of the new toxic property that is involved in the mechanism of causation of the disease. Preparation and characterization of these human mutant ALS CuZn SOD proteins for the purpose of identifying their disease-causing properties is a major objective of this project. Studies of another copper protein, stellacyanin, are designed to test the hypothesis that this blue copper protein is a cell wall protein that is involved in plant defense mechanisms. Another human protein, blood coagulation factor VIII, is involved in causing hemophilia A. It possesses protein sequences characteristic of blue copper binding sites, but no structural information nor characteristics of these sites are yet known. The copper-binding domains of this protein will be expressed, purified, and characterized. A major objective of this research is to understand the role of copper in human coagulation factor VIII and how it is related to the overall function of this protein in the blood clotting process. Finally, new metalloproteins will be designed, based on CuZn SOD and stellacyanin. They will be prepared using site-directed mutagenesis, expressed, and purified and their new properties will be characterized.
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