Veenstra T D, Gross M D, Hunziker W, Kumar R
Department of Medicine, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
J Biol Chem. 1995 Dec 22;270(51):30353-8. doi: 10.1074/jbc.270.51.30353.
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.
钙结合蛋白D28K每摩尔蛋白质结合3摩尔铽。为了确定该蛋白质六个EF-手型结构中哪一个负责铽的结合，我们构建了该蛋白质的三种突变形式，一种缺失EF-手型2（RCaBP δ2），另一种缺失EF-手型2和6（RCaBP δ2,6），第三种仅包含EF-手型3和4（RCaBP δ1,2,5,6），并通过荧光技术检测它们的结合特性。全长钙结合蛋白D28K、RCaBP δ2和RCaBP δ2,6每摩尔蛋白质以高亲和力结合3摩尔铽。因此，EF-手型结构域2和6对于蛋白质与钙的结合并非必需，并且缺失EF-手型2和/或6不会改变铽与蛋白质的结合模式。利用色氨酸残基的共振能量转移，每个蛋白质的一个高亲和力铽结合位点（位点A）比其他两个位点（位点B和C）具有更高的亲和力。位点A在其他两个位点之前被填满。钙竞争实验表明，从位点A置换铽所需的钙量比从位点B或C置换铽所需的钙量更多。从铽到钬的能量转移实验表明，两个铽结合位点彼此靠近，而第三个位点与其他两个位点距离较远。为了确定EF-手型3或4是否负责铽的结合，我们检测了δ1,2,5,6 RCaBP构建体的铽结合特性。截短的蛋白质RCaBP δ1,2,5,6包含一个铽结合位点。对铽与RCaBP δ1,2,5,6构建体结合的分析表明，位点4结合铽，而位点3不结合。对蛋白质铽结合特性的分析表明，大鼠脑钙结合蛋白D28K的EF-手型1、4和5负责铽的结合。
