ASSIGNMENT OF THE N-15 NMR-SPECTRA OF REDUCED AND OXIDIZED ESCHERICHIA-COLI THIOREDOXIN

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the N-15 NMR spectra of reduced and oxidized Escherichia coli thioredoxin (M(r) 12000) uniformly labeled with N-15 has been performed. The N-15 chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using N-15-H-1 correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear H-1 spectra. The differences in the N-15 chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.