Kägi J H, Vasák M, Lerch K, Gilg D E, Hunziker P, Bernhard W R, Good M
Environ Health Perspect. 1984 Mar;54:93-103. doi: 10.1289/ehp.54-1568188.
All mammalian metallothioneins characterized contain a single polypeptide chain of 61 amino acid residues, among them 20 cysteines providing the ligands for seven metal-binding sites. Native metallothioneins are usually heterogeneous in metal composition, with Zn, Cd, and Cu occurring in varying proportions. However, forms containing only a single metal species, i.e., Zn, Cd, Ni, Co, Hg, Pb, Bi, have now been prepared by in vitro reconstitution from the metal-free apoprotein. By spectroscopic analysis of such derivatives it was established that all cysteine residues participate in metal binding, that each metal ion is bound to four thiolate ligands, and that the symmetry of each complex is close to that of a tetrahedron. To satisfy the requirements of the overall Me7(Cys-)20 stoichiometry, the complexes must be combined to form metal-thiolate cluster structures. Experimental proof for the occurrence of such clusters comes from the demonstration of metal-metal interactions by spectroscopic and magnetic means. Thus, in Co(II)7-metallothionein, the Co(II)-specific ESR signals are effectively suppressed by antiferromagnetic coupling of juxtaposed paramagnetic metal ions. By monitoring changes in ESR signal size occurring on stepwise incorporation of Co(II) into the protein, it is possible to follow the building up of the clusters. This process is biphasic. Up to binding of four equivalents of Co(II), the ESR amplitude increases in proportion to the metal content, indicating generation of magnetically noninteracting high-spin complexes. However, upon addition of the remaining three equivalents of Co(II), these features are progressively suppressed, signaling the formation of clusters. The same mode of cluster formation has also been documented for Cd and Hg. The actual spatial organization of the clusters and the polypeptide chain remains to be established. An attractive possibility is the arrangement of the tetrahedral metal-thiolates in adamantane-like structures surrounded by properly folded segments of the chain providing the ligands. 1H-NMR data and infrared absorption measurements are consistent with a tightly folded structure rich in beta-type conformation.
所有已被鉴定的哺乳动物金属硫蛋白都含有一条由61个氨基酸残基组成的单链多肽，其中20个半胱氨酸为7个金属结合位点提供配体。天然金属硫蛋白的金属组成通常是异质的，锌、镉和铜以不同比例存在。然而，现在已经通过从无金属的脱辅基蛋白进行体外重组制备出了仅含单一金属种类（即锌、镉、镍、钴、汞、铅、铋）的形式。通过对此类衍生物的光谱分析确定，所有半胱氨酸残基都参与金属结合，每个金属离子与四个硫醇盐配体结合，并且每个配合物的对称性接近四面体。为了满足整体Me7(Cys-)20化学计量的要求，这些配合物必须结合形成金属硫醇盐簇结构。这种簇的存在的实验证据来自于通过光谱和磁性手段证明的金属 - 金属相互作用。因此，在Co(II)7 - 金属硫蛋白中，相邻顺磁性金属离子的反铁磁耦合有效地抑制了Co(II)特异性的电子自旋共振（ESR）信号。通过监测在将Co(II)逐步掺入蛋白质过程中ESR信号大小的变化，可以追踪簇的形成过程。这个过程是双相的。在结合四个当量的Co(II)之前，ESR幅度与金属含量成比例增加，表明生成了磁性非相互作用的高自旋配合物。然而，在加入剩余的三个当量的Co(II)后，这些特征逐渐受到抑制，表明簇的形成。对于镉和汞也记录到了相同的簇形成模式。簇和多肽链的实际空间组织仍有待确定。一种有吸引力的可能性是四面体金属硫醇盐以金刚烷样结构排列，周围是提供配体的链的适当折叠片段。1H - NMR数据和红外吸收测量结果与富含β型构象的紧密折叠结构一致。
