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Metal-dependent protein folding mechanisms of the alpha domain of human metallothionein.

机译：人金属硫蛋白的α结构域的金属依赖性蛋白折叠机制。

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摘要

Over 30% of all proteins require metal ions for functional or structural purposes. The effect of metal-protein interactions on the stability of the folded protein structure and the role that the metal ion(s) play in the folding mechanism has been largely overlooked. Metallothionein (MT) is a unique metalloprotein that folds in the presence of divalent metal ions (M) to form two distinct domains, each containing a metal-thiolate cluster with stoichiometries of M3(Scys)9 (beta domain) and M4(S cys)11 (alpha domain), respectively, making it an ideal model to study metal-induced folding mechanisms. The present study investigates the metal binding reactivity of the isolated alpha domain of human MT-1a with Cd2+ using the techniques of CD, UV absorption, NMR and stopped-flow spectroscopy, ESI mass spectrometry and molecular dynamics/mechanics computational techniques.;From the results of these studies, it is proposed that the metal-free protein can adopt two different structural motifs: (1) a well-defined H-bonded structure that is induced by the template effect of the previously coordinated metal-thiolate cluster, or (2) a random conformation that is associated with the de nova synthesized protein. Furthermore, the metallation mechanism is predicted to he dependent on the initial structure of the apo protein.;Keywords: Metallothionein, metal-dependent folding, cooperativity, cadmium, CD spectroscopy, ESI mass spectrometry, stopped-flow kinetics, 113 Cd NMR spectroscopy, molecular mechanics, molecular dynamics.;The results of the metallation studies were as follows: (1) the rate of the metallation reaction of Cd2+ was shown to be very fast (kobs = 106 108 M-1 s-1) when the demetallated protein was the reacting species. (2) a reduction in the metallation rate was observed in the presence of a chemical denaturant. (3) the MM3/MD calculations of the demetallated protein domains showed significant retention of the backbone structural features imposed by the metal-thiolate cluster. (4) the MM3/MD calculations of the in silico-synthesized metal-free protein as a linear strand showed random behaviour of the polypeptide backbone. (5) partially-metallated domain intermediates were detected in the presence of sub-stoichiometric Cd2+ concentrations confirming that metal binding proceeds by four sequential, non-cooperative metallation reactions, and (6) excess Cd2+ was shown to induce the formation of a novel Cd5alpha species in which the fifth Cd2+ ion inserted into the cluster by coordination to two cysteinyl sulfur ligands.
机译：为了功能或结构目的，所有蛋白质中超过30％需要金属离子。金属-蛋白质相互作用对折叠的蛋白质结构的稳定性的影响以及金属离子在折叠机制中的作用已被大大忽略。金属硫蛋白（MT）是一种独特的金属蛋白，可在二价金属离子（M）存在的情况下折叠形成两个不同的域，每个域均包含金属硫醇盐簇，其化学计量比为M3（Scys）9（β域）和M4（S cys） ）11（α域），使其成为研究金属诱导的折叠机制的理想模型。本研究使用CD，UV吸收，NMR和停止流光谱，ESI质谱和分子动力学/力学计算技术研究了人类MT-1a的分离的α结构域与Cd2 +的金属结合反应性。这些研究的结果表明，无金属蛋白可以采用两种不同的结构基序：（1）由先前配位的金属-硫醇盐簇的模板效应诱导的明确定义的H键结构，或（ 2）与新合成蛋白质相关的随机构象。此外，预测金属化机理取决于载脂蛋白的初始结构。关键词：金属硫蛋白，金属依赖性折叠，协同作用，镉，CD光谱，ESI质谱，停流动力学，113 Cd NMR光谱，分子力学，分子动力学；金属化研究的结果如下：（1）当脱金属蛋白质时，Cd2 +的金属化反应速率非常快（kobs = 106 108 M-1 s-1）。是反应物种。 （2）在化学变性剂的存在下观察到金属化速率的降低。 （3）脱金属蛋白结构域的MM3 / MD计算表明，金属硫醇盐簇强加了骨架结构特征。 （4）以硅链合成的无金属蛋白质为线性链的MM3 / MD计算显示了多肽主链的随机行为。 （5）在亚化学计量的Cd2 +浓度存在下检测到部分金属化的结构域中间体，证实金属结合是通过四个连续的非合作金属化反应而进行的，并且（6）过量的Cd2 +已显示出诱导了新型Cd5alpha的形成物种，其中第五个Cd2 +离子通过与两个半胱氨酰硫配体配位而插入簇中。

著录项

  • 作者

    Rigby Duncan, Kelly E.;

  • 作者单位

    The University of Western Ontario (Canada).;

  • 授予单位 The University of Western Ontario (Canada).;
  • 学科 Chemistry Inorganic.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 203 p.
  • 总页数 203
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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