We have shown that CD2 is an excellent scaffold protein [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] . It retains its native structure following insertion of the EF-hand motif both in the absence and presence of Ca 2+ ions. This provides the foundation for measuring the intrinsic Ca 2+ binding affinity with minimized contribution of protein conformational change. In addition, the aromatic Grafting approach to probe site-specific metal binding properties of Ca 2+ -binding proteins. (a) Schematic representation of the grafting approach. Any predicted linear Ca 2+ -binding sequence can be inserted into the host protein CD2 domain 1 (CD2.D1) between residues S52 and G53 without disrupting the integrity of the host protein. Metal binding to the engineered protein can be monitored by taking advantage of a potential LRET pair, the buried Trp (W32) within the two layers of beta-sheets and the terbium ion bound to the inserted sequence. (b) Flow chart showing the application of grafting approach to confirm metal binding of predicted Ca 2+ binding sites. Adapted with permission from ref. [23, 29] . residues in CD2 enable us to obtain Tb 3+ affinity of the grafted Ca 2+ binding loop using FRET. Ca 2+ and its analog La 3+ are able to compete with Tb 3+ for the grafted metal binding site. We have also optimized the length of two glycine linkers that connect the Ca 2+ binding loop and CD2 to provide sufficient freedom for the loop. The grafted EFloop III of CaM in different protein environments and scaffolds (such as CD2) has similar metal binding affinities for La 3+ and Tb 3+ , which implies that the grafted EF-hand loop is largely solvated and functions independently from the host protein or the protein environment. More importantly, using high resolution NMR and 15 N labeled protein, we have shown that both Ca 2+ and La 3+ specifically interact with the residues in the grafted EF-loop [25] , suggesting that the grafted loop retains its native Ca 2+ binding property. In addition, to dissect the contribution of the EF-loop and its flanking segments on Ca 2+ affinity, we have inserted the EF-loop, the loop with the exiting F-helix, and the loop with both EF-helices of Site III of CaM into CD2. In contrast to the largely unfolded structure of the isolated peptide fragment, the inserted flanking helices are partially formed, as revealed by both CD and NMR. Ca 2+ affinity is enhanced about 3-10 fold when the flanking helices are attached. Further, we have first estimated the intrinsic Ca 2+ affinities of the four EF-hand loops of CaM (I-IV) by individually grafting them into CD2. EF-loop I exhibits the strongest while EF-loop IV has the weakest binding affinity for Ca 2+ , La 3+ , and Tb 3+ . EF-loops I-IV of CaM have dissociation constants for Ca 2+ of 34, 245, 185, and 814 μM, respectively. Based on the results, we proposed a charge-ligandbalanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are major determinants for the Ca 2+ binding affinities of EFloops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca 2+ binding properties and to estimate the cooperativity and conformational change contributions of coupled EF-hand motifs. We have shown that the contribution of the cooperativity and conformational change to the Ca 2+ affinity for the C-terminal is 40% greater than that for the N-terminal. The same approach will be used to probe the site-specific Ca 2+ affinity of bacterial proteins. Furthermore, we have applied high resolution pulsed-fieldgradient diffusion NMR (PFG NMR) and analytical ultracentrifugation to investigate the oligomeric state of the isolated EF-loop III of CaM in CD2 with and without the flanking helices. The loop without the helices (CaM-CD2-III-5G) remains unpaired in solution in the absence and presence of Ca 2+ . However, the loop with the flanking helices (CaM-CD2-III-5G-EF) is a dimer in the presence of Ca 2+ [34] . Our findings suggest that hydrophobic residues on flanking helices play an essential role in dimerization and coupling of two EF-hand motifs for stronger Ca 2+ affinity.