Structure and function of metal-binding human metallothionein

哺乳动物金属硫蛋白（MT）将7个二价金属离子与其20个半胱氨酸结合。它作为锌的细胞储存库和锌的分布，虽然被认为参与重金属的解毒和抗氧化防御。MT是研究与细胞中金属处理有关的分子相互作用的合适模型。然而，由于MT的不稳定性，微生物中功能性MT蛋白的生产受到限制。因此，用于研究的MT供应继续依赖于从哺乳动物组织中纯化，特别是因为镉诱导MT的动物肝脏可以制备相当大量的MT。I)我们开发了MT作为一种间质融合蛋白的过剩生产系统，通过亲和层析纯化，用DTT孵育裂解蛋白，用镉或锌重组硫蛋白制备。2)为了提高二聚体和四聚体MT的结合能力和稳定性，我们设计了二聚体和四聚体MT的基因，并成功地在大肠杆菌中过表达，生成了功能性低聚体MT。3)砷是一种有毒元素，存在于大气中，也存在于定量和陆地环境中。我们通过紫外吸收光谱、ICP-AES和MALDI-TOF-MS证明了超过6克的AS^<3+>原子与一个人MT分子结合。4)我们在人MT-2中构建了一个新的金属结合位点，利用该蛋白作为支架研究金属结合的结构和功能。为了评估突变蛋白的金属结合亲和力，我们对野生型和突变蛋白进行了pH滴定。5)我们发现野生型和突变型mt均能结合DNA，但两种mt对DNA的结合亲和力不同。

Mammalian metallothionein (MT) binds 7 divalent metal ions to its 20 cysteines. It has a role as a cellular reservoir for zinc and in distribution of zinc and is although thought to participate in detoxification of heavy metals and in antioxidant defenses. MT is a suitable model for investigating molecular interactions relating to the handling of metals in cells. However, the production of functional MT proteins in microoganisms has been limited because of the instability of MT. The supply of MT for research, therefore, continues to rely on its purification from mammalian tissue, in particular because rather large quantities can be prepared from livers of animals in which MT has been induced by cadmium.I) We developed the overproduction system for MT as an intein fusion protein, purified by affinity chromatography, cleaved proteolytically by incubation with DTT, and prepared by reconstitution of thionein with cadmium or zinc.2) To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MTs and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs.3) Arsenic is a toxic element that is found in the atmosphere, as well as in quantic and terrestrial environments. We have demonstrated that more than 6 gram atoms of AS^<3+> bind one molecule of human MT by UV absorption spectroscopy, ICP-AES, and MALDI-TOF-MS.4) We have constructed a new metal-binding site in the human MT-2, using the protein as a scaffold to investigate the structure and function of metal-binding. To evaluate the metal-binding affinity of the mutant proteins, we performed pH titrations of wild-type and mutant proteins.5) We found that wild-type and mutant MTs bound DNA but the two MTs showed different affinity of binding to DNA.