Calmodulin (CaM) is a trigger calcium-dependent protein that regulates many biological processes. We have successfully engineered a series of model proteins, each containing a single EF-hand loop but with increasing numbers of Gly residues linking the EF-hand loop to a scaffold protein, cluster of differentiation 2 (CD2), to obtain the site-specific calcium-binding ability of a protein with EF-hand motifs without the interference of cooperativity. Loop III of calmodulin with two Gly linkers in CD2 (CaM-CD2-III-5G) has metal affinities with K(d) values of 1.86 x 10(-4) and 5.8 x 10(-5) M for calcium and lanthanum, respectively. The oligomeric states of the CD2 variants were examined by pulsed-field-gradient nuclear magnetic resonance (PFG NMR). The diffusion coefficient values of CD2 variants are about 11.1 x 10(-7) cm(2)/s both in the presence and absence of metal ions, which are the same as that of wild-type CD2. This suggests that the isolated EF-loop III of calmodulin inserted in the scaffold protein is able to bind calcium and lanthanum as a monomer, which is in contrast to the previous observation of the EF-hand motif. Our results imply that additional factors that reside outside of the EF-loop III may contribute to the pairing of EF-hand motifs of calmodulin. This result is of interest as it opens up the way for studying the ion-binding properties of isolated EF-hands, which in turn can answer important questions about the properties of EF-hands, the large and important group of calcium-binding signaling proteins.