Department of Chemical Biology, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a, 50-383 Wrocław, Poland.
Metallomics. 2023 Oct 4;15(10). doi: 10.1093/mtomcs/mfad061.
Metallothioneins (MTs) are small, Cys-rich proteins present in various but not all organisms, from bacteria to humans. They participate in zinc and copper metabolism, toxic metals detoxification, and protection against reactive species. Structurally, they contain one or multiple domains, capable of binding a variable number of metal ions. For experimental convenience, biochemical characterization of MTs is mainly performed on Cd(II)-loaded proteins, frequently omitting or limiting Zn(II) binding features and related functions. Here, by choosing 10 MTs with relatively well-characterized structures from animals, plants, and bacteria, we focused on poorly investigated Zn(II)-to-protein affinities, stability-structure relations, and the speciation of individual complexes. For that purpose, MTs were characterized in terms of stoichiometry, pH-dependent Zn(II) binding, and competition with chromogenic and fluorescent probes. To shed more light on protein folding and its relation with Zn(II) affinity, reactivity of variously Zn(II)-loaded MTs was studied by (5,5'-dithiobis(2-nitrobenzoic acid) oxidation in the presence of mild chelators. The results show that animal and plant MTs, despite their architectural differences, demonstrate the same affinities to Zn(II), varying from nano- to low picomolar range. Bacterial MTs bind Zn(II) more tightly but, importantly, with different affinities from low picomolar to low femtomolar range. The presence of weak, moderate, and tight zinc sites is related to the folding mechanisms and internal electrostatic interactions. Differentiated affinities of all MTs define their zinc buffering capacity required for Zn(II) donation and acceptance at various free Zn(II) concentrations (pZn levels). The data demonstrate critical roles of individual Zn(II)-depleted MT species in zinc buffering processes.
金属硫蛋白（MTs）是存在于各种生物体（从细菌到人类）中的富含半胱氨酸的小蛋白。它们参与锌和铜代谢、有毒金属解毒以及对活性物质的保护。在结构上，它们包含一个或多个结构域，能够结合可变数量的金属离子。出于实验方便，MTs 的生化特性主要在 Cd(II)负载的蛋白质上进行研究，通常忽略或限制 Zn(II)结合特性和相关功能。在这里，我们选择了来自动物、植物和细菌的 10 种结构相对较好的 MTs，重点研究了 Zn(II)与蛋白质亲和力、稳定性-结构关系以及单个配合物的形态。为此，MTs 的化学计量比、pH 依赖性 Zn(II)结合以及与显色和荧光探针的竞争情况进行了特征描述。为了更深入地了解蛋白质折叠及其与 Zn(II)亲和力的关系，通过在温和螯合剂存在下用（5,5'-二硫代双（2-硝基苯甲酸）氧化法研究了各种 Zn(II)负载 MTs 的反应性。结果表明，尽管动物和植物 MTs 在结构上存在差异，但它们对 Zn(II)的亲和力相同，范围从纳摩尔到低皮摩尔。细菌 MTs 结合 Zn(II)的亲和力更强，但重要的是，其亲和力范围从低皮摩尔到低飞摩尔。弱、中、强锌结合位点的存在与折叠机制和内部静电相互作用有关。所有 MTs 的不同亲和力定义了它们在各种游离 Zn(II)浓度（pZn 水平）下的锌缓冲能力，这是 Zn(II)供体和受体所必需的。这些数据表明了个体 Zn(II)耗尽的 MT 物种在锌缓冲过程中发挥着关键作用。
