A functional and degenerate pair of EF hands contains the very high affinity calcium-binding site of calbindin-D_28K

Calbindin-D_28K, a member of the troponin C superfamily of calcium-binding proteins, had six putative EF hand domains containing one very high affinity and two to three lower affinity calcium-binding sites. The location and binding activity of the calcium-binding sites were examined with a recombinant calbindin-D_28K protein. This protein (Calb I-II) only contained EF hand domains 1 and 2 of calbindin-D_28K. Binding of calcium and calcium analogs, the lanthanides, by the recombinant protein was determined in fluorescence emission experiments. Calb I-II bound 1 mol of terbium/mol of protein. Terbium was displaced from Calb I-II by other lanthanides and calcium. Fluorescence from terbium was not quenched by holmium. These results and Hill plots of calcium binding activity, determined from intrinsic protein fluorescence measurements, indicated the presence of a single high affinity calcium-binding site on Calb I-II. The properties of the binding site indicated that the very high affinity site of calbindin-D_28K was located in EF hand domains 1 and 2 of the protein. In addition, these findings indicated the NH₂-terminal pair of EF hands in calbindin-D_28K did not depend on interactions with other domains in the protein for high affinity calcium binding activity. The results suggested at least one calcium-binding domain of calbindin-D_28K can exist as an independent EF hand pair.