Huang Zhong Xian, Gao Yuan, Yu Wen Hao, Zhang Su Yan, Yang Peng Yuan
Chemical Biology Lab, Department of Chemistry, Fudan University, 200433, Shanghai, China.
J Inorg Biochem. 2002 Nov 25;92(3-4):183-92. doi: 10.1016/s0162-0134(02)00492-0.
The differences in metal-thiolate coordination and reactivity of mammalian metallothionein (MT) domains are closely related to their distinct, highly conservative cysteine number and position. Monkey metallothionein-1, containing a beta-domain with Cd(3)S(9) cluster and an alpha-domain with Cd(4)S(11) cluster, was used to evaluate the role of cysteine residues in the formation of MT's metal-thiolate clusters. The possible influence of cysteine residues on the binding and stability of MT domains has been examined with the metallothionein mutants: N4C, T27C and N4C/T27C, which possess ten or eleven cysteine residues in the re-constructed beta-domain, respectively. Assisted by study of UV, CD and electrospray ionization mass spectroscopy (ESI-MS) and their reactivity with DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), we found that besides the original alpha-domain, some kinds of new domain containing 4-cadmium-thiolate clusters were formed in the N4C and N4C/T27C mutants of mkMT1. These new domains displayed metal binding and kinetic reactivity with DTNB similar to the alpha-domain. However, the thermal stability of the mutants was less stable than that of WT mkMT1. This might result from the disturbance of the inter-domains hydrogen bonds and of the non-cysteine amino acid residue arrangement.
哺乳动物金属硫蛋白（MT）结构域中金属硫醇盐配位和反应性的差异与其独特的、高度保守的半胱氨酸数量和位置密切相关。猴金属硫蛋白-1含有一个具有Cd(3)S(9)簇的β结构域和一个具有Cd(4)S(11)簇的α结构域，用于评估半胱氨酸残基在MT金属硫醇盐簇形成中的作用。通过金属硫蛋白突变体N4C、T27C和N4C/T27C研究了半胱氨酸残基对MT结构域结合和稳定性的可能影响，这些突变体在重建的β结构域中分别具有十个或十一个半胱氨酸残基。通过紫外光谱、圆二色光谱和电喷雾电离质谱（ESI-MS）研究以及它们与5,5'-二硫代双（2-硝基苯甲酸）（DTNB）的反应性，我们发现除了原始的α结构域外，mkMT1的N4C和N4C/T27C突变体中形成了一些含有4-镉硫醇盐簇的新结构域。这些新结构域表现出与α结构域相似的金属结合和与DTNB的动力学反应性。然而，突变体的热稳定性低于野生型mkMT1。这可能是由于结构域间氢键和非半胱氨酸氨基酸残基排列受到干扰所致。
