Conformational transitions of thioredoxin in guanidine hydrochloride

Abstract

Spectral and hydrodynamic measurements of thioredoxin from E. coli indicate that the compact globular structure of the native protein is significantly unfolded in the presence of guanidine hydrochloride concentrations in excess of 3.3 M at neutral pH and 25.degree. C. This conformational transition having a midpoint at 2.5 M denaturant is quantitatively reversible and highly cooperative. Stopped-flow measurements of unfolding in 4 M denaturant, observed with tryptophan fluorescence as the spectral probe, reveal a single kinetic phase having a relaxation time of 7.1 .+-. 0.2 s. Refolding measurements in 2 M denaturant reveal 3 kinetic phases having relaxation times of 0.54 .+-. 0.23, 14 .+-. 6 and 500 .+-. 130 s, accounting for 12 .+-. 2%, 10 .+-. 1% and 78 .+-. 3% of the observed change in tryptophan fluorescence. The dominant slowest phase is generated in the denatured state with a relaxation time of 42 s observed in 4 M denaturant. Both the slowest phase observed in refolding and the generation of the slowest phase in the denatued state have an activation enthalpy of 22 .+-. 1 kcal/mol. These features of the slowest phase are compatible with an obligatory peptide isomerizarion of proline-76 to its cis isomer prior to refolding.