Biosynthetic 15N and 13C isotope labelling of glutathione in the mixed disulfide with Escherichia coli glutaredoxin documented by sequence-specific NMR assignments.

Abstract

A biosynthetic procedure for obtaining 13C-15N doubly labelled glutathione from readily available precursor molecules is described. Isolation of the mutant Escherichia coli [C14S]glutaredoxin from E. coli cultures grown on 15N-13C doubly labelled media in the absence of reducing agents yields the mixed disulfide labelled in both the protein and the glutathione. 15N NMR assignments for glutathione obtained from two-dimensional [15N,1H]-correlation spectroscopy (COSY), and 13C NMR assignments for the entire mixed disulfide obtained from combined use of three-dimensional ct-HA[CAN]HN experiments and HCCH-total correlation spectroscopy ([HCCH]-TOCSY) demonstrated unequivocally that the glutathione is uniformly labelled with both 15N and 13C. This result also supports earlier suggestions that the intracellular glutaredoxin activity is sensitive to the glutathione redox status of the cell. Complete sets of 1H, 13C and 15N chemical shifts of both components in the mixed disulfide of [C14S]glutaredoxin and glutathione were obtained from the sequence-specific NMR assignments.