Calcium regulates many biological processes through interaction with proteins with different conformational, dynamic, and metal binding properties. Previous studies have shown that the electrostatic environment plays a key role in calcium binding affinity. In this research, we aim to dissect the contribution of the electrostatic environment to calcium binding affinity using protein design. Many natural calcium binding proteins undergo large conformational changes upon calcium binding which hampers the study of these proteins. In addition, cooperativity between multiple calcium binding sites makes it difficult to study site-specific binding affinity. The design of a single calcium binding site into a host system eliminates the difficulties that occur in the study of calcium binding affinity. Using a computer algorithm we have rationally designed several calcium binding sites with a pentagonal bipyramidal geometry in the non-calcium dependent cell adhesion protein CD2 (CD2-D1) to better investigate the key factors that affect calcium binding affinity. The first generation proteins are all in varying electrostatic environments. The conformational and metal binding properties of each of these designed proteins were analyzed. The second generation designed protein, CD2.6D79, was designed based on criteria learned from the first generation proteins. This protein contains a novel calcium binding site with ligands all from the â-strands of the non-calcium dependent cell adhesion protein CD2. The resulting protein maintains native secondary and tertiary packing and folding properties. In addition to its selectivity for calcium over other mono and divalent metal ions, it displays strong metal binding affinities for calcium and its analogues terbium and lanthanum. Furthermore, our designed protein binds CD48, the ligand binding partner of CD2, with an affinity three-fold stronger than CD2. The electrostatic potential of the calcium binding site was modified through mutation to facilitate the study of the effect of electrostatic interactions on calcium binding affinity. Several charge distribution mutants display varying metal binding affinities based on their charge, distance to the calcium binding site, and protein stability. This study will provide insight into the key site factors that control calcium binding affinity and calcium dependent biological function.