Identification of metal binding sites in rat brain calcium-binding protein

Calbindin D-28K binds 3 mol of terbium per mol of protein. To determine which of six EF hand structures in the protein are responsible for terbium binding, we constructed three mutant forms of this protein, one lacking EF-hand 2 (RCaBP Delta 2), the other lacking EF-hands 2 and 6 (RCaBP Delta 2,6), and the third containing only EF-hands 3 and 4 (RCaBP Delta 1,2,5,6), and examined their binding properties by fluorescence techniques, Full-length calbindin D-28K and RCaBP Delta 2 and RCaBP Delta 2,6 bound 3 mol of terbium per mol of protein with high affinity. Thus, EP hand domains 2 and 6 are not essential for calcium binding to the proteins, and an absence of EF-hands 2 and/or 6 does not alter the pattern of terbium binding to the protein. Using resonance energy transfer from tryptophan residues, one of the high affinity terbium-binding sites (site A) had a greater affinity than the other two sites (sites B and C) of each protein. Site A was filled before the other two sites. Calcium competition experiments showed that a greater amount of calcium was required to displace terbium from site A than from sites B or C, Energy transfer experiments from terbium to holmium showed that two of the terbium-binding sites are in close proximity while the third site is distant from the other two sites, To determine whether EF-hand 3 or 4 was responsible for binding of terbium, we examined the terbium binding properties of a Delta 1,2,5,6 RCaBP construct. The truncated protein RCaBP Delta 1,2,5,6 contained a single terbium-binding site, Analysis of the terbium binding to RCaBP Delta 1,2,5,6 construct showed that site 4 bound terbium, whereas site 3 did not. Analysis of the terbium binding characteristics of the proteins suggests that EF-hands 1, 4, and 5 of rat brain calbindin D-28K are responsible for terbium binding.