Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry

Abstract

Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-ultraviolet (UV) circular dichroism (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and its Cys32-alkylated derivatives in 1% acetic acid (pH 2.7). Thermal unfolding of oxidized (Oxi) and reduced (Red) -thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX), which are derivatives of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchange profiles, and near-UV CD appear to support a two-state folding/unfolding model. Minor mass peaks in the H/D exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process. The relative stabilities as measured by melting temperatures by both CD and mass spectral charge states are, Oxi-TRX, GS-ethyl-TRX, Cys-ethyl-TRX, and Red-TRX. The introduction of the Cys-32-ethylglutathionyl group provides extra stabilization that results from additional hydrogen bonding interactions between the ethylglutathionyl group and the protein. Near-UV CD data show that the local environment near the active site is perturbed to almost an identical degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-bonding ethylcysteinyl group. Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX.

Fig. 1.

Simulated structure of GS-ethyl-TRX derived from the crystal structure of Oxi-TRX (Kim et al. 2001b). The glutathionyl group is represented in the ball and stick format (sulfur in yellow, nitrogen in blue, carbon in grey, oxygen in red). The TRX protein is represented as ribbons (α-helices in red, β-sheets in yellow, and turns and loops in blue and grey). The dotted lines indicate two salt bridges and three hydrogen bonds induced by the ethylglutathionyl group.

Fig. 2.

Electrospray ionization mass spectra of Oxi-TRX in 1% acetic acid at different temperatures: (A) 85°C, (B) 65°C, and (C) 25°C. Ion peaks at m/z 1668, 1460, and 1298 representing charge states 7+, 8+, and 9+, respectively, were attributed to the folded form, F. Ion peaks at m/z 1168, 1062, 973, 899, 835, and 779 representing charge states 10+ to 15+ were attributed to the unfolded form, U.

Fig. 3.

Heat denaturation curves of TRXs in 1% acetic acid deducted from the temperature-dependent, charge-state distributions obtained by electrospray ionization mass spectrometry. T_ms for Oxi-, GS-ethyl-, Cys-ethyl-, and Red-TRX are 67, 56, 54, and 53°C, respectively.

Fig. 4.

Near ultraviolet circular dichroism spectra of thermal denaturation of oxidized Escherichia coli thioredoxin (34 μM in 1% acetic acid).

Fig. 5.

Denaturation curves of Escherichia coli thioredoxins in 1% acetic acid obtained from the analyses of near ultraviolet circular dichroism spectra. Each data point is from the intensity measured at 280 nm. T_ms for Oxi-, GS-ethyl-, Cys-ethyl-, and Red-TRX are 61, 53, 49, and 43°C, respectively.

Fig. 6.

Evolution of the eightfold charged ion peaks of Escherichia coli thioredoxins during on-line hydrogen/deuterium (H/D) exchange-in experiments in 1% AcOD/D₂O at 50°C. Time points refer to H/D exchange periods. Dashed peaks were obtained by mass spectral measurements of the fully deuterated proteins.

Fig. 7.

Temperature-dependent alteration of the hydorgen/deuterium exchange mass profiles. Comparison of the eightfold charged ion peak of Escherichia coli thioredoxins at different temperatures after an exchange-in period of 105±10 sec in 1% AcOD/D₂O.

Fig. 8.

Deuterium incorporation in Escherichia coli thioredoxins with time (A) total deuterium content and (B) after back exchange of side-chain deuteriums.