Identification of metal-binding sites in rat brain calcium-binding protein.
Calbindin D28K 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 D28K and RCaBP delta 2 and RCaBP delta 2,6 bound 3 mol of terbium per mol of protein with high affinity. Thus, EF-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 D28K are responsible for terbium binding.
Veenstra
T D
TD
Department of Medicine, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Gross
M D
MD
Hunziker
W
W
Kumar
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D064092:Calbindin 1; D064026:Calbindins; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004735:Energy Transfer; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000378:metabolism*; D014364:Tryptophan
C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D013725:Terbium; D014364:Tryptophan