Chan Jayna, Huang Zuyun, Watt Ian, Kille Peter, Stillman Martin
Department of Chemistry, The University of Ontario, London, Ontario, Canada.
Chemistry. 2008;14(25):7579-93. doi: 10.1002/chem.200800787.
The ubiquitous protein metallothionein (MT) has proven to be a major player not only in the homeostasis of Cu(I) and Zn(II), but also binds all the Group 11 and 12 metals. Metallothioneins are characterised by the presence of numerous cys-x-cys and cys-cys motifs in the sequence and are found naturally with either one domain or two, linked, metal-binding domains. The use of chains of these metal-thiolate domains offers the possibility of creating chemically tuneable and, therefore, chemically dependent electrochemical or photochemical surface modifiers or as nanomachinery with nanomechanical properties. In this work, the metal-binding properties of the Cd(4)-containing domain of alpha-rhMT1a assembled into chains of two and three concatenated domains, that is, "necklaces", have been studied by spectrometric techniques, and the interactions within the structures modelled and interpreted by using molecular dynamics. These chains are metallated with 4, 8 or 12 Cd(II) ions to the 11, 22, and 33 cysteinyl sulfur atoms in the alpha-rhMT1a, alphaalpha-rhMT1a, and alphaalphaalpha-rhMT1a proteins, respectively. The effect of pH on the folding of each protein was studied by ESI-MS and optical spectroscopy. MM3/MD simulations were carried out over a period of up to 500 ps by using force-field parameters based on the reported structural data. These calculations provide novel information about the motion of the clustered metallated, partially demetallated, and metal-free peptide chains, with special interest in the region of the metal-binding site. The MD energy/time trajectory conformations show for the first time the flexibility of the metal-sulfur clusters and the bound amino acid chains. We report unexpected and very different sizes for the metallated and demetallated proteins from the combination of experimental data, with molecular dynamics simulations.
普遍存在的蛋白质金属硫蛋白（MT）已被证明不仅在Cu（I）和Zn（II）的体内平衡中起主要作用，而且还能结合所有第11族和第12族金属。金属硫蛋白的特征是序列中存在大量的cys-x-cys和cys-cys基序，并且天然存在一个或两个相连的金属结合结构域。使用这些金属硫醇盐结构域链提供了创建化学可调谐的、因此化学依赖性的电化学或光化学表面改性剂或作为具有纳米机械性能的纳米机械的可能性。在这项工作中，通过光谱技术研究了组装成两个和三个串联结构域链（即“项链”）的α-rhMT1a含Cd(4)结构域的金属结合特性，并使用分子动力学对结构内的相互作用进行了建模和解释。这些链分别用4、8或12个Cd(II)离子与α-rhMT1a、αα-rhMT1a和ααα-rhMT1a蛋白中的11、22和33个半胱氨酰硫原子进行金属化。通过电喷雾电离质谱（ESI-MS）和光谱学研究了pH对每种蛋白质折叠的影响。使用基于报道的结构数据的力场参数进行了长达500皮秒的MM3/MD模拟。这些计算提供了关于聚集的金属化、部分脱金属化和无金属肽链运动的新信息，特别关注金属结合位点区域。MD能量/时间轨迹构象首次展示了金属-硫簇和结合的氨基酸链的灵活性。我们通过实验数据与分子动力学模拟相结合，报告了金属化和脱金属化蛋白质出人意料且非常不同的大小。
