The goal of this project is to design and analyze calcium-binding proteins in order to gain an understanding of key site factors, such as ligand type and charge, that control calcium-binding affinity. Our long-term goal is to understand the mechanism of calcium-modulated signaling and cell adhesion. Since isotope-labeled metal ions can be tracked by radiological, NMR or chemical means, our success in designing metal binding Sites into arbitrary proteins will likely lead to new ways of developing useful reagents for diagnostic tests and chemotherapy. We hypothesize that the frequency of each residue used as a ligand in known calcium-binding sites represents its relative calcium binding ability. Both negatively charged residues and the side chain rigidity of the carboxyl group determine the order for calcium affinity: Asp> Glu> Asn, Ser, Thr> Gln. Our novel approach allows us to minimize global effects from the overall protein conformation by keeping the same protein structure with only a few residues changed. In this case since protein environment is maintained, the measured calcium binding affinity can be directly correlated to the local structural features of the metal-binding sites. The contribution of the residue type (rotamer), charge distribution at the ligand positions, and bond length to the affinity will be measured. To help us design calcium-binding sites in proteins and predict their calcium-binding affinity, we also propose to dissect the contribution of charge distribution to calcium affinity by introducing charged residues around the calcium sites with different electrostatic arrangements. We further propose to characterize the Ca(II) binding sites (ligand types and the geometry) using high resolution methods. A comparison of the calcium binding sites in proteins determined by high-resolution methods with those of the originally designed target will be carried out to optimize our methods for design of calcium