To overcome the above-mentioned barriers and limitations associated with naturally-occurring Ca 2+ binding proteins, we have developed a grafting approach for engineering a single Ca 2+ -binding site in order to dissect the key structural factors that control Ca 2+ -binding affinity, conformational change and cooperativity. In principle, the key determinants for Ca 2+ affinity can be systematically introduced into a stable host protein frame and evaluated by eliminating or minimizing the contribution of conformational change. The key factors that are essential for Ca 2+ -dependent conformational change can be further revealed by analyzing the folding, stability, dynamics, and conformations of the host protein upon binding of a designed Ca 2+ -binding site without the complication of cooperativity. The cooperativity of two-coupled Ca 2+ -binding sites can then be estimated once the intrinsic Ca 2+ -binding affinities of both sites are obtained based on the energetics relationship. Figure 3 shows our grafting approach in obtaining site-specific calcium binding affinity using domain1 CD2 as a scaffold protein.