Probing site-specific calmodulin calcium and lanthanide affinity by grafting
Probing site-specific calmodulin calcium and lanthanide affinity by grafting
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DOI：
10.1021/ja042786x
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发表时间：
2005-03-23
影响因子：
15
通讯作者：
Yang, JJ
中科院分区：
文献类型：
--
作者：
Ye, YM;Lee, HW;Yang, JJ
钙调素（CaM）作为一种触发/感受蛋白，在钙信号级联反应中起着重要的生物学作用。理解Ca 2+信号传导机制的一个挑战是获得EF-手蛋白，特别是CaM的单个Ca 2+结合位点的Ca 2+结合特性的位点特异性信息。在本文中，我们报告的第一个估计的内在钙离子亲和力的四个EF-手循环的钙调蛋白（I-IV）通过单独移植到域1的CD 2。利用宿主蛋白中的Trp残基，我们首先使用Tb 3+芳香族共振能量转移确定了所有四个接枝EF-环对Tb 3+、Ca 2+和La 3+的金属结合亲和力。EF-环I对Ca 2+、La 3+和Tb 3+表现出最强的结合亲和力，而EF-环IV具有最弱的金属结合亲和力。EF-环I-IV的钙调素的解离常数为34，245，185，和814 μ M，分别与顺序I > III接近II > IV的钙。这些研究结果支持一个电荷配体平衡的模型，其中带负电荷的配体残基的数量和平衡的静电齿齿排斥相邻的带电残基是两个主要的决定因素的相对Ca 2+结合的亲和力EF-环钙调素。我们的嫁接方法提供了一种新的策略，以获得特定位点的Ca 2+结合特性和更好的估计耦合EF-手蛋白的协同性和构象变化的贡献。
Ca2+ binding is essential for the biological functions of calmodulin (CaM) as a trigger/sensor protein to regulate many biological processes in the Ca2+-signaling cascade. A challenge in understanding the mechanism of Ca2+ signaling is to obtain site-specific information about the Ca2+ binding properties of individual Ca2+-binding sites of EF-hand proteins, especially for CaM. In this paper, we report the first estimation of the intrinsic Ca2+ affinities of the four EF-hand loops of calmoduin (I-IV) by individually grafting into the domain 1 of CD2. Taking advantage of the Trp residues in the host protein, we first determined metal-binding affinities for Tb3+, Ca2+, and La3+ for all four grafted EF-loops using Tb3+ aromatic resonance energy transfer. EF-loop I exhibits the strongest binding affinity for Ca2+, La3+, and Tb3+, while EF-loop IV has the weakest metal-binding affinity. EF-loops I-IV of CaM have dissociation constants for Ca2+ of 34, 245, 185, and 814 mu M, respectively, with the order I > III approximate to II > IV. These findings support a charge-ligand-balanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are two major determinants for the relative Ca2+-binding affinities of EF-loops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca2+ binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.