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

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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.