To establish an approach to obtain the site-specific calcium binding affinity of EF-hand proteins, we have successfully designed a series of model proteins, each containing the EF-hand calcium-binding loop 3 of calmodulin, but with increasing numbers of Gly residues linking the loop to domain 1 of CD2. Structural analyses, using different spectroscopic methods, have shown that the host protein is able to retain its native structure after insertion of the 12-residue calcium-binding loop and retains a native thermal stability and thermal unfolding behavior. In addition, calcium binding to the engineered CD2 variants does not result in a significant change from native CD2 conformation. The CD2 variant with two Gly linkers has been shown to have the strongest metal binding affinity to Ca(II) and La(III). These experimental results are consistent with our molecular modeling studies, which suggest that this protein with the engineered EF-loop has a calmodulin-like calcium binding geometry and backbone conformation. The addition of two Gly linkers increases the flexibility of the inserted EF-loop 3 from calmodulin, which is essential for the proper binding of metal ions.