77SE NMR STUDIES OF THIOREDOXIN LABELED W/ L [3 77SE] SELENOCYSTEINE

SIR提供(1)Selenocystine；50 mg[92-94%-77 硒半胱氨酸；100毫克(L)-碲胱氨酸；150毫克 一种细菌表达系统，允许高效替代 硒半胱氨酸在蛋白质中的半胱氨酸残基(见Miller，S.， 首页--期刊主要分类--期刊细介绍--期刊题录与文摘--期刊详细文摘内容 3404-3412)。它涉及到各自基因的过度表达 半胱氨酸营养缺陷菌中T7启动子/聚合系统的辅助作用 紧张。应用该体系替代了两种半胱氨酸 大肠杆菌中硫氧还蛋白的残留量。分离得到(Se)-2-硫氧还蛋白 具有生化特征的。利用这种方法，L-[3-77Se]半胱氨酸 由SIR提供的将并入硫氧还蛋白，它将 用77Se富集型二硒化桥取代二硫键。 分离的(77Se)2-硫氧还蛋白将用77Se核磁共振进行研究 光谱学。硫氧还蛋白中的二硫化物是一个双电子 氧化还原载体，用于将核苷酸还原为脱氧核苷酸。 我们将开发一种直接确定氧化还原状态的方法 用77Se核磁共振波谱研究蛋白质中的二硫化物。

The SIR provided (1) Selenocystine; 50mg [92-94% - 77 Se2]Selenocystine; 100mg (l)-tellurocystine; 150mg We have developed a bacterial expression system which allows the efficient substitution of cysteine residues in a protein by selenocysteine (see: Miller, S., Senn, H., Gsell, B., Vetter, W. and Bvck, A. (1994), Biochemistry 33, 3404-3412). It involves overexpression of the respective gene with the aid of the T7 promoter/polymerize system in a cysteine auxotroph strain. The system was applied to substitute the two cysteine residues in E.coli thioredoxin. (Se)2-Thioredoxin was isolated and biochemically characterized. Using this method, L-[3-77Se]cysteine provided by SIR will be incorporated into thioredoxin which will replace the disulfide bridge by a 77Se-enriched diselenide bridge. The isolated (77Se)2-Thioredoxin will be studied by 77Se NMR spectroscopy. The disuffide in thioredoxin serves as a two electron redox carrier for the reduction of nucleotides to deoxy nucleotides. We will develop a method directly determine the redox status of disulfides in proteins using 77Se NMR spectroscopy.