Snyder E E, Buoscio B W, Falke J J
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1990 Apr 24;29(16):3937-43. doi: 10.1021/bi00468a021.
The molecular mechanisms by which protein Ca(II) sites selectively bind Ca(II) even in the presence of high concentrations of other metals, particularly Na(I), K(I), and Mg(II), have not been fully described. The single Ca(II) site of the Escherichia coli receptor for D-galactose and D-glucose (GGR) is structurally related to the eukaryotic EF-hand Ca(II) sites and is ideally suited as a model for understanding the structural and electrostatic basis of Ca(II) specificity. Metal binding to the bacterial site was monitored by a Tb(III) phosphorescence assay: Ca(II) in the site was replaced with Tb(III), which was then selectively excited by energy transfer from protein tryptophans. Photons emitted from the bound Tb(III) enabled specific detection of this substrate; for other metals binding was detected by competitive displacement of Tb(III). Representative spherical metal ions from groups IA, IIA, and IIIA and the lanthanides were chosen to study the effects of metal ion size and charge on the affinity of metal binding. A dissociation constant was measured for each metal, yielding a range of KD's spanning over 6 orders of magnitude. Monovalent metal ions of group IA exhibited very low affinities. Divalent group IIA metal ions exhibited affinities related to their size, with optimal binding at an effective ionic radius between those of Mg(II) (0.81 A) and Ca(II) (1.06 A). Trivalent metal ions of group IIIA and the lanthanides also exhibited size-dependent affinities, with an optimal effective ionic radius between those of Sc(III) (0.81 A) and Yb(III) (0.925 A). The results indicate that the GGR site selects metal ions on the basis of both charge and size.(ABSTRACT TRUNCATED AT 250 WORDS)
即使在存在高浓度其他金属（特别是Na(I)、K(I)和Mg(II)）的情况下，蛋白质Ca(II)位点仍能选择性结合Ca(II)的分子机制尚未得到充分描述。大肠杆菌D-半乳糖和D-葡萄糖受体（GGR）的单个Ca(II)位点在结构上与真核生物的EF-手型Ca(II)位点相关，非常适合作为理解Ca(II)特异性的结构和静电基础的模型。通过Tb(III)磷光测定法监测金属与细菌位点的结合：位点中的Ca(II)被Tb(III)取代，然后通过蛋白质色氨酸的能量转移选择性激发Tb(III)。结合的Tb(III)发射的光子能够特异性检测该底物；对于其他金属，通过Tb(III)的竞争性取代检测结合情况。选择IA族、IIA族和IIIA族的代表性球形金属离子以及镧系元素来研究金属离子大小和电荷对金属结合亲和力的影响。测量了每种金属的解离常数，得到的KD范围跨越6个数量级。IA族的单价金属离子表现出非常低的亲和力。IIA族的二价金属离子表现出与其大小相关的亲和力，在Mg(II)（0.81 Å）和Ca(II)（1.06 Å）之间的有效离子半径处具有最佳结合。IIIA族的三价金属离子和镧系元素也表现出大小依赖性亲和力，在Sc(III)（0.81 Å）和Yb(III)（0.925 Å）之间具有最佳有效离子半径。结果表明，GGR位点基于电荷和大小来选择金属离子。（摘要截断于250字）
