This program project brings together a group of investigators for the purpose of studying, in concert, metalloprotein structure and design. The incorporation of metal binding sites in proteins significantly expands the biochemical repertoire available to polypeptides. Metalloprotein design, the predetermined placement and alteration of metal centers in proteins, would be a powerful addition to our current capabilities of protein engineering. Such design will require an improved comprehension of the structural and chemical basis for the affinity, specificity and function of metal sites, as it pertains to the folding and assembly, stability, regulation, and chemical activity of metalloproteins. We will pursue four basic themes central to metalloprotein structure and design: (1) folding and assembly of proteins directed by structural metal site(s) (Projects I, III, VI); (2) regulation of activity or binding properties by the structural changes from metal binding (Projects I, II, IV-VI, VIII); (3) introduction of a metal with a desired geometry and metal specificity (Projects II, IV- VIII); and (4) transplantation of a metal site with a specified activity (Projects IV, VI-VIII). The focus of the individual projects extends from prediction to experimental testing of the predictions. Each project supplies and/or contributes significantly to the identification of the structural and chemical factors that are most important in metal site design. Together these projects will investigate the major types of metal sites in proteins (Fe, di-Fe, Fe in heme and FeS clusters, Cu, Zn, Mn, and Ca sites) and their results will be highly synergistic for understanding common and variable features of metalloprotein structure and function. The Chazin laboratory will apply NMR to defining Ca and Zn binding in calcyclin. Site-directed mutagenesis by the Stout laboratory will probe the Fe-S cluster of aconitase. The Ghadiri laboratory will use chemical and genetic approaches to study metal- assisted folding and stability of peptides and proteins. The Goodin and McRee groups will collaborate on remodeling the cytochrome c peroxidase heme pocket with the Goodin laboratory creating and characterizing new An, Mn and Cu metalloproteins, and the McRee laboratory evaluating their crystal structures. The Tainer laboratory will focus on structure-based redesign and crystallography of the di-Fe sites in ribonucleotide reductase and myohemerythrin and the Mn site in human mitochondrial superoxide dismutase. The computational efforts of the Noodleman laboratory will define the structural basis for Fe, Mn, Cu, and Zn binding and activity in superoxide dismutases, metalloantibodies, and other systems. The Getzoff and Gascoigne laboratories will make and determine atomic structures for metalloantibodies designed using the structural results from all these projects, with an emphasis on Zn, Cu, Mn, Ca, and Fe binding sites. Overall, we seek to develop, test, and apply rules governing metal site coordination, affinity and activity in proteins, by using both theory and experiment. The ability to understand and ultimately control the binding and activity of protein metal sites, which are required by about one-third of all proteins for stability and function, is of great medical and scientific importance.