We propose here to design, construct, and characterize, both functionally and structurally, novel metalloantibodies. The results from the seven other projects on the structural and chemical roles of metal sites in the folding and assembly, stability, regulation, and chemical activity of metalloproteins will contribute experimental and computational information for improved metalloantibody design. This iterative approach will allow us to rigorously test and refine our understanding of the requirements for functional metal sites in proteins. We chose the murine monoclonal anti-cytochrome c antibody E8 as the scaffold for our redesign experiments. This E8 antibody binds its natural metalloprotein antigen with high affinity (10-9-10-10M), providing the potential for studies of electronic coupling between engineered metal sites in the antibody and the naturally occurring heme- Fe in cytochrome c. The E8 antibody has been well characterized biochemically, immunologically, and structurally: the gene sequences of the E8 variable domains, and the crystallographic structures of both the free antigen-binding fragment (Fab) and the Fab/cytochrome c complex have been determined in the Getzoff laboratory, and a system for secreting mutant antibodies in a transformed cell line that expresses the wild-type E8 light chain paired with mutant E8 heavy chains has been developed in the Gascoigne laboratory. Toward the ultimate goal of creating a site-specific Zn protease, we have transplanted a Zn site into the heavy chain of the E8 antibody by site- directed mutagenesis. Besides proteolytic activity, design targets include redox reactions and metal-mediated binding of cytochrome c. For the resulting site-directed mutant metalloantibodies, our aims are to characterize metal-binding properties, measure antigen-binding and potential catalytic activities, and determine and analyze three- dimensional crystallographic structures. The long term objective of this proposal is to engineer scientifically and medically useful metalloantibodies that synergistically combine the remarkable binding and recognition properties of antibodies with the powerful functional and catalytic properties of metalloproteins.
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