COMPARISON OF THE HYDROGEN-EXCHANGE BEHAVIOR OF REDUCED AND OXIDIZED ESCHERICHIA-COLI THIOREDOXIN

Hydrogen-deuterium exchange rates for the amide protons in oxidized (d isulfide) and reduced (dithiol) thioredoxin have been measured using a series of N-15-H-1 HSQC spectra at various times after buffer exchang e into 99% (H2O)-H-2. Information on exchange rates and protection fac tors was obtained for both forms of thioredoxin for 68 amide protons u sing this method; in general, the rates obtained by this method were f or amide protons of residues in the hydrogen-bonded beta-sheet and alp ha-helix secondary structure of thioredoxin. Estimates of the exchange rate for those amide protons that exchanged with rates too fast to me asure 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 t he loops adjacent to it (residues 73-75 and 91-98). Amide proton excha nge rates provide a qualitative estimate of the backbone mobility, and the differences in hydrogen exchange behavior between the two forms o f thioredoxin are consistent with those observed in calculations of po lypeptide chain dynamics obtained from N-15 relaxation measurements [S tone, 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, c onsistent with their very close similarity in structure and backbone d ynamics. Significant differences in behavior are observed in the activ e site sequence and in the regions of the protein that are close to th is sequence in the three-dimensional structure, including portions of the beta-strand and alpha-helical sequences immediately adjacent to th e active site. In particular, the exchange rate of the Cys 35 amide pr oton is significantly slowed for oxidized thioredoxin compared with th e reduced form. In general, the amide proton exchange rates are consis tently lower for the oxidized form of thioredoxin, in accord with lite rature reports of lower mobility of the polypeptide backbone in the vi cinity of the active site compared with the reduced protein, The rathe r generalized loosening of the protein structure upon reduction, indic ated by the hydrogen exchange measurements, provides further evidence that the functional differences observed between the two forms of thio redoxin can most likely be ascribed to the greater flexibility of the reduced form of the protein.