Tryptophan Fluorescence Study of Conformational Transitions of the Oxidized and Reduced Form of Thioredoxin

Arne Holmgren

doi:10.1016/s0021-9258(19)45481-1

Abstract

Abstract Fluorescence emission spectra of the oxidized and reduced forms of thioredoxin (thioredoxin-S2 and thioredoxin-(SH)2, respectively) from Escherichia coli were determined at varying pH, solvent composition, and concentration of urea or guanidine hydrochloride. Thioredoxin-S2, with only 2 tryptophan residues at positions 28 and 31, showed a strongly quenched tryptophan fluorescence (Q = 0.02) which remained constant from pH 2 to pH 10. Addition of ethanol or dioxan up to 50% (v/v) at pH 7.0 did not perturb the tryptophan emission. Guanidine hydrochloride from 2 to 4 m increased the fluorescence intensity of thioredoxin-S2 3-fold and the emission maximum was shifted from 342 nm to 355 nm indicating unfolding of the protein. The beginning of this reversible transition was also observed in 6 m urea. Between 4 to 7 m guanidine only a small increase in tryptophan fluorescence occurred, indicating that thioredoxin-S2 was largely unfolded. Thioredoxin-(SH)2, obtained by chemical reduction of thioredoxin-S2 by dithiothreitol, showed a pronounced pH dependence of fluorescence with a maximum at pH 5, where the emission intensity was 6.5-fold higher than that of thioredoxin-S2. The fluorescence intensity decreased on the alkaline side of pH 5 and seemed to fit a titration curve with an apparent pK of 6.75. Between 0 to 2 m guanidine hydrochloride the fluorescence intensity for thioredoxin-(SH)2 increased almost 2-fold in a transition not observed with thioredoxin-S2. Between 2 to 4 m guanidine the fluorescence intensity decreased in a transition similar to the increase in intensity for thioredoxin-S2. Under all experimental conditions, the tryptophan fluorescence intensity of thioredoxin-(SH)2 was higher than that of thioredoxin-S2, suggesting that the disulfide bond of thioredoxin-S2 was an important quenching group. The fluorescence emission spectrum of yeast thioredoxin, which has a single tryptophan residue in a position homologous to Trp-31 of E. coli thioredoxin, showed a low quantum yield, (Q = 0.03), of tryptophan fluorescence in its oxidized form. Reduction of the disulfide bond increased the fluorescence intensity 1.2-fold. This suggests that the conformational change which accompanies reduction of thioredoxin-S2 from E. coli has its main effect on the emission of Trp-28.

Highlights

Sometimes rather subtle changes in the conformation of a protein give rise to fairly large changes in the fluorescence characteristics, whereas other physical methods such as optical rotatory dispersion or ultracentrifupation fail to show any change. This is the case with thioredoxin from Escherichia coli where chemical or enzymatical reduction of the oxidized form results in a a-fold increase in the quantum yield of tryptophan fluorescence indicating a localized conformational change [7]
This paper describes studies of the ultraviolet fluorescence properties of thioredoxin from E. coli with the aim of obtaining information about the structure surrounding the tryptophan residues close to the active site of thioredoxinS
Fluorescence Emission Spectra of Thioredoxin-Fluorescence emission spectra of native thioredoxin-Sz and thioredoxin-(SH)t at pH 7.0 after excitation at 280 nm are shown in Fig. 1. -1s observed previously [7], the spectra were dominated by tryptophan emission with a maximum at 342 nm and the fluorescence intensity was more than S-fold higher for thioredoxin-(SH)2

Summary

Introduction

Between 0 to 2 M guanidine hydrochloride the fluorescence intensity for thioredoxin-(SH)2 increased almost 2-fold in a transition not observed with thioredoxin-S2. This is the case with thioredoxin from Escherichia coli where chemical or enzymatical reduction of the oxidized form results in a a-fold increase in the quantum yield of tryptophan fluorescence indicating a localized conformational change [7].

Results

Conclusion