Next, we grafted two predicted 29-residue EF-hand motifs, one from the Shr of S. pyrogenes (CD2.Shr.EF) and the viral nsP1 of Sindbis virus (CD2.Sin.EF), into CD2.D1 to examine their Ca 2+ binding capability by using aromatic residue-sensitized Tb 3+ luminescence resonance energy transfer (Tb 3+ -LRET) ( Figure 5 ). Circular dichroism studies of both engineered proteins showed a notable trough at ~216 nm which is characteristic of β-sheet structure. More negative signals were observed below 240 nm due to the contribution from the insertion of the helix-loop-helix sequences. Both proteins were able to bind the Ca 2+ analog, Tb 3+ , with affinities of 25.1 μM for CD2.Shr.EF and 16.4 μM for CD2.Sin.EF ( Figure 5(c)-(d) ). The biological relevance of these EF-hand Ca 2+ -binding motifs will be further investigated.