2-DIMENSIONAL H-1 NUCLEAR-MAGNETIC-RESONANCE STUDIES OF THE HALF-SATURATED (CA2+)1 STATE OF CALBINDIN D(9K) - FURTHER IMPLICATIONS FOR THE MOLECULAR-BASIS OF COOPERATIVE CA2+ BINDING
2-DIMENSIONAL H-1 NUCLEAR-MAGNETIC-RESONANCE STUDIES OF THE HALF-SATURATED (CA2+)1 STATE OF CALBINDIN D(9K) - FURTHER IMPLICATIONS FOR THE MOLECULAR-BASIS OF COOPERATIVE CA2+ BINDING
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DOI：
10.1006/jmbi.1993.1291
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发表时间：
1993-05-20
影响因子：
5.6
通讯作者：
CHAZIN, WJ
中科院分区：
文献类型：
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作者：
CARLSTROM, G;CHAZIN, WJ
Calbindin D9k表现出两个钙离子的协同结合，因此研究蛋白质的半饱和状态对于理解结合过程是至关重要的。然而，在平衡条件下，半饱和态的布居并不明显。为了避免这个问题，C-末端结合位点(II位)上的一个绝对保守的谷氨酸残基被突变为谷氨酰胺(E65Q)，导致钙亲和力显著降低，并允许对Calbindin D9k进行详细的二维1H核磁共振分析，其中一个钙离子只结合在N端的EF-手中。获得了(Ca~(2+))_1E65Q的完全1H共振指认，以及apo和(Ca~(2+))_2的近全指认。通过分析与钙滴定反应的化学位移变化，确定了Ⅱ位的钙结合常数为1·1(±0·2)×10~3m-1。从核Overhaser效应、主链自旋-自旋耦合常数和主链酰胺质子的交换率确定了二级结构和全局折叠模式的元素。虽然突变对蛋白质的二级结构和整体折叠的影响很小，但它极大地降低了C-末端对钙的亲和力，以至于(钙)2E65Q不对应于标准的(钙)2状态。从半饱和状态的分析可以明显看出，在载脂蛋白→(Ca~(2+))_1(I)→(Ca~(2+))_2结合途径的每一步，Calbindin D9k确实发生了一些结构重组和内部动力学变化。当第一个离子结合到N端的EF-Hand上时，分子的那一半采取了类似于完全钙负载的蛋白质状态的构象和动态，而在C端的EF-Hand中只发生了很小的变化。只有当第二个钙离子结合时，C-末端EF-Hand才会切换到完全钙负载状态。结合早期对载脂蛋白→(Ca~(2+))_1(II)→(Ca~(2+))_2结合途径的四个研究结果，这些发现表明与Ca~(2+)结合相关的蛋白质构象和动力学的变化有助于协同观察，并且两种结合途径协同结合事件的分子细节是不同的。
Calbindin D9kexhibits cooperative binding of two calcium ions, hence study of the half-saturated states of the protein is critical to understanding the binding process. However, the half-saturated states are not significantly populated under equilibrium conditions. To circumvent this problem, an absolutely conserved glutamic acid residue in the C-terminal binding site (site II) has been mutated to glutamine (E65Q), causing a substantial reduction in calcium affinity and permitting detailed two-dimensional1H NMR analysis of calbindin D9kwith a calcium ion bound only in the N-terminal EF-hand. Complete1H resonance assignments have been obtained for (Ca2+)1E65Q, as well as near complete assignments for the apo and (Ca2+)2states. A value of 1·1 (±0·2) × 103M-1has been determined for the calcium binding constant in site II, from an analysis of the chemical shift changes in response to titration with calcium. The elements of secondary structure and global folding patterns were identified from nuclear Overhauser effects, backbone spin-spin coupling constants and the exchange rates of backbone amide protons. Although the mutation has only very small effects on the secondary structure and global fold of the protein, it so drastically lowers affinity for Ca2+in the C-terminal site that (Ca2+)2E65Q does not correspond to a standard (Ca2+)2state. From the analysis of the half-saturated state, it is apparent that some reorganization of the structure and changes in the internal dynamics of calbindin D9kdoes occur for each step of the apo → (Ca2+)1(I) → (Ca2+)2binding pathway. When the first ion is bound to the N-terminal EF-hand, that half of the molecule adopts a conformation and dynamic state similar to the fully calcium-loaded protein state, whereas only minor change occur in the C-terminal EF-hand. It is only upon binding of the second calcium ion that the C-terminal EF-hand switches over to the fully calcium-loaded state. Together with the results from four earlier study of the apo → (Ca2+)1(II) → (Ca2+)2binding pathway, these findings indicate that changes in protein conformation and dynamics associated with Ca2+binding contribute to the observed cooperatively, and that the molecular details of the cooperative binding events are different for the two binding pathways.