Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding.

The conformational transition observed upon addition of guanidine hydrochloride (Gdn-HCl) to solutions of oxidized Escherichia coli thioredoxin is dominated by a slow kinetic phase (time constant tau 1 = 300-800 s) that has features appropriate to a proline peptide isomerization. This observation has been interpreted as reflecting a folding pathway involving an obligatory isomerization of the imide peptide bond between isoleucine-75 and proline-76 [Kelley, R. F., & Stellwagen, E. (1984) Biochemistry 23, 5095-5102]; this peptide bond is known to have the cis configuration in the folded state [Eklund, H., Cambillan, C., Sjöberg, B.-M., Holmgren, A., Jörnvall, H., Höög, J.-O., & Brändén, C.-I. (1984) EMBO J. 3, 1443-1449]. We have tested this hypothesis by examining the conformational transitions of two thioredoxin mutants, trxA76 having an alanine substituted for proline-76 and trxA2 [Russel, M., & Model, P. (1983) J. Bacteriol. 154, 1064-1070] having proline-34 replaced with serine. Both mutant proteins display far-ultraviolet circular dichroic spectra similar to that of native wild-type thioredoxin. The tryptophan fluorescence emission of native trxA2 is equivalent to that of wild-type thioredoxin, while the emission intensity of native trxA76 at 350 nm is 2-fold greater. Tryptophan fluorescence and peptide ellipticity measurements indicate that the mutant proteins undergo two-state and reversible equilibrium unfolding transitions upon addition of guanidine hydrochloride (Gdn-HCl). These transitions are centered at 2.4 and 1.5 M Gdn-HCl for trxA2 and trxA76, respectively, as compared to a midpoint of 2.5 M denaturant for wild-type thioredoxin. As observed for wild-type thioredoxin, fluorescence measurements reveal monophasic unfolding kinetics for trxA2 at a variety of final denaturant concentrations. The tau for unfolding varies monotonically from 210 s in 2.4 M Gdn-HCl to 7 s for a final Gdn-HCl concentration of 3.5 M. Refolding of denatured trxA2 in 1.5 M Gdn-HCl detected by fluorescence measurements is described by three kinetic phases with time constants and fractional amplitudes (alpha) similar to those of wild-type thioredoxins. The fractional amplitude (alpha 1) of the slowest of these phases, tau 1 = 430 +/- 38 s in 2.0 M Gdn-HCl, decreases with final Gdn-HCl concentration. Multimixing experiments suggest that this phase results from an equilibration between denatured forms and has a tau of 34 s in 4 M denaturant, features previously observed for the wild-type protein.(ABSTRACT TRUNCATED AT 400 WORDS)