Comparison of the hydrogen-exchange behavior of reduced and oxidized Escherichia coli thioredoxin.

Hydrogen-deuterium exchange rates for the amide protons in oxidized (disulfide) and reduced (dithiol) thioredoxin have been measured using a series of 15N-1H HSQC spectra at various times after buffer exchange into 99% 2H2O. Information on exchange rates and protection factors was obtained for both forms of thioredoxin for 68 amide protons using this method; in general, the rates obtained by this method were for amide protons of residues in the hydrogen-bonded beta-sheet and alpha-helix secondary structure of thioredoxin. Estimates of the exchange rate for those amide protons that exchanged with rates too fast to measure by hydrogen--deuterium exchange were made by saturation-transfer measurements, which were particularly useful in defining the hydrogen exchange behavior of the active site Cys-Gly-Pro-Cys sequence and of the loops adjacent to it (residues 73-75 and 91-98). Amide proton exchange rates provide a qualitative estimate of the backbone mobility, and the differences in hydrogen exchange behavior between the two forms of thioredoxin are consistent with those observed in calculations of polypeptide chain dynamics obtained from 15N relaxation measurements [Stone, M. J., et al. (1993) Biochemistry 32, 426-435]. For most of the protein, the exchange rates are close to identical in the two forms, consistent with their very close similarity in structure and backbone dynamics. Significant differences in behavior are observed in the active site sequence and in the regions of the protein that are close to this sequence in the three-dimensional structure, including portions of the beta-strand and alpha-helical sequences immediately adjacent to the active site.(ABSTRACT TRUNCATED AT 250 WORDS)