EFFECT OF DISULFIDE BRIDGE FORMATION ON THE NMR-SPECTRUM OF A PROTEIN- STUDIES ON OXIDIZED AND REDUCED ESCHERICHIA-COLI THIOREDOXIN

As a prelude to complete structure calculations of both the oxidized a nd reduced forms of Escherichia coli thioredoxin (M(r) 11700), we have analyzed the NMR data obtained for the two proteins under identical c onditions. The complete aliphatic C-13 assignments for both oxidized a nd reduced thioredoxin are reported. Correlations previously noted bet ween C-13 chemical shifts and secondary structure are confirmed in thi s work, and significant differences are observed in the C-beta and C g amma shifts between cis- and trans-proline, consistent with previous w ork that identifies this as a simple and unambiguous method of identif ying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys(32)-Gly-Pro-Cys(35) sequence causes changes in the H-1, N-15 and C-13 chemical shifts of residues close to the activ e site, some of them quite far distant in the amino acid sequence. Cou pling constants, both backbone and side chain, show some differences b etween the two proteins, and the NOE connectivities and chemical shift s are consistent with small changes in the positions of several side c hains, including the two tryptophan rings (Trp(28) and Trp(31)). These results show that, consistent with the biochemical behavior of thiore doxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.