Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.
Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.
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使用（113）CD NMR在金属蛋白中探测天然金属结合位点：概述。
DOI：
10.1007/978-94-007-5179-8_6
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Reilly B
中科院分区：
文献类型：
--
作者：
Armitage IM;Drakenberg T;Reilly B
我们的实验室多年来一直在这一领域积极发表文章，本章的目的是提供尽可能全面的概述。在简要回顾与 113Cd NMR 方法相关的基本原理之后，我们将介绍对金属蛋白的 113Cd 化学位移进行彻底文献检索的结果。本章中更新的 113Cd 化学位移图将进一步说明 113Cd 化学位移与配位配体（N、O、S）的性质和配位数/几何形状的良好相关性，重申该方法不仅可以用于识别未表征情况下蛋白质配体的性质，还可以用于识别金属结合位点的动态。具体例子将来自对碱性磷酸酶、Ca2+结合蛋白和金属硫蛋白的研究。就大肠杆菌碱性磷酸酶而言，这是一种二聚锌金属酶，共有 6 个金属离子（每个单体 3 个）直接或间接参与为酶提供最大催化活性和结构稳定性，113Cd NMR 与 13C 和 31P NMR 方法相结合，有助于分离出各类金属结合位点的功能。也许最重要的是，这些研究揭示了这种酶在金属缺乏条件下负协同作用的化学基础。同样值得注意的是，这些 NMR 研究先于 X 射线晶体结构的出现。就钙结合蛋白而言，我们将重点关注两种蛋白质：钙结合蛋白 D9k 和钙调蛋白。对于钙结合蛋白 D9k 及其突变体，113Cd NMR 可用于跟踪金属结合位点的实际变化和金属结合的协同性。配体与钙调蛋白的结合已通过 113Cd NMR 进行了广泛研究，表明金属结合位点并不直接参与配体结合。然而，113Cd 的化学位移对金属离子环境的微小变化非常敏感。就金属硫蛋白而言，我们将反思 113Cd 取代以及通过质子检测异核 1H-113Cd 多量子相干方法 (HMQC) 建立特异性 Cd 与 Cys 残基连接性对于金属硫蛋白 3D 结构的初始建立至关重要，金属硫蛋白是缺乏 α 螺旋和 β 片层常规二级结构元件的蛋白质家族，也是第一个用结合 Cd 鉴定的天然蛋白质。 113Cd NMR 研究还能够表征各个位点对 113Cd 的亲和力，以及在竞争实验中对其他二价金属离子（Zn、Cu 和 Hg）的亲和力。
Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with 113Cd NMR methods, we will present the results from a thorough literature search for 113Cd chemical shifts from metalloproteins. The updated 113Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the 113Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca2+ binding proteins, and metallothioneins. In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, 113Cd NMR, in conjunction with 13C and 31P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceeded the availability of the X-ray crystal structure. In the case of the calcium binding proteins, we will focus on two proteins: calbindin D9k and calmodulin. For calbindin D9k and its mutants, 113Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with 113Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The 113Cd chemical shifts are, however, exquisitely sensitive to minute changes in the metal ion environment. In the case of metallothionein, we will reflect upon how 113Cd substitution and the establishment of specific Cd to Cys residue connectivity by proton-detected heteronuclear 1H-113Cd multiple-quantum coherence methods (HMQC) was essential for the initial establishment of the 3D structure of metallothioneins, a protein family deficient in the regular secondary structural elements of α-helix and β-sheet and the first native protein identified with bound Cd. The 113Cd NMR studies also enabled the characterization of the affinity of the individual sites for 113Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.