Preparation and 113Cd NMR studies of homogeneous reconstituted metallothionein: reaffirmation of the two-cluster arrangement of metals.
Preparation and 113Cd NMR studies of homogeneous reconstituted metallothionein: reaffirmation of the two-cluster arrangement of metals.
复制标题
均相重构金属硫蛋白的制备和 113Cd NMR 研究：重申金属的双簇排列。
DOI：
10.1021/bi00345a001
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Wehrli,S
中科院分区：
文献类型：
--
作者：
Otvos,JD;Engeseth,HR;Wehrli,S
Department of Chemistry，UniversityofWisconsin-密尔沃基，密尔沃基，威斯康星州53201接收于1985年6月19日摘要：在所有7个结合位点用113 Cd重构的兔肝金属硫蛋白2的113 Cd NMR分析先前表明金属排列成两个金属-硫醇盐簇[Otvos，J.D.，& Armitage，I. M.等人（1980）Proc. Acad. Sci. USA 77，7094-7098]。然而，蛋白质的光谱总是包含七个以上的共振，这表明样品在某种程度上是异质的。以不同方式重构但也给出具有多于七个共振的光谱的U3 Cd金属硫蛋白的最近研究的结果已被解释为反对金属结合的两簇模型，并支持其中蛋白质的结构柔性允许簇的许多构型亚态共存的模型[Vasak，M.，霍克斯，通用电气，尼科尔森，J. K.，& Sadler，PJ（1985）Biochemistry 24，740-747]，这里提供的数据表明，作为金属重构的副产物形成的金属硫蛋白的二聚体和较大的寡聚体是这些工作者归因于构型底物的至少一些113 Cd共振的可能来源。通过凝胶过滤去除污染的低聚物产生可验证的均质蛋白质，其113 Cd光谱由7个强度相当的共振组成。通过二维化学位移相关光谱和对单个共振的113 Cd-113 Cd分裂模式的光谱模拟，对先前提出的两个金属硫醇盐簇的存在和结构进行了无敌意的确认。富含半胱氨酸的金属结合蛋白，被认为主要参与抵抗重金属毒性和锌和铜的体内平衡（Nordberg & Kojima，1979; Webb & Cain，1982;布雷迪，1982）。为了与这些假定的功能保持一致，MT有能力绑定一个
Department of Chemistry, University of Wisconsin—Milwaukee, Milwaukee, Wisconsin 53201 Received June 19, 1985 abstract: 113Cd NMR analysis of rabbit liver metallothionein 2 reconstituted with 113Cd at all seven binding sites has previously indicated that the metals are arranged in two metal-thiolate clusters [Otvos, J. D., & Armitage, I. M.(1980) Proc. Natl. Acad. Sci. USA 77, 7094-7098]. Spectra of the protein always contained more than seven resonances, however, suggesting the samples were in some way heterogeneous. Results of a recent study of U3Cd metallothionein reconstituted in a different manner but also giving spectra with more than seven resonances have been interpreted as arguing against the two-cluster model of metal binding and in favor of a model in which structural flexibility of the protein allows many configurational substates of the cluster (s) to coexist [Vasak, M., Hawkes, GE, Nicholson, J. K., & Sadler, PJ (1985) Biochemistry 24, 740-747], Data are presented here that indicate that dimersand larger oligomers of metallothionein formed as byproducts of metal reconstitution are the likely source of at least some of the 113Cd resonances attributed by these workers to configurational substrates. Removal of the contaminating oligomers by gel filtration yields a verifiably homogeneous protein whose 113Cd spectrum consists of seven resonances of comparable intensity. Unambiguous confirmation of the existence and structures of the two previously proposed metal-thiolate clusters was obtained by two-dimensional chemicalshift correlation spectroscopy and spectral simulation of the 113Cd-113Cd splitting patterns of the individual resonances.IN^ etallothioneins (MTs) 1 comprise a unique family of low molecular weight, cysteine-rich metal binding proteins thought to be centrally involved in resistance to heavy-metal toxicity and the homeostasis of zinc and copper (Nordberg & Kojima, 1979; Webb & Cain, 1982; Brady, 1982). In keeping with these postulatedfunctions, MT has the capacity to bind an