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1 Department of Chemistry, Oregon State University, Corvallis, Oregon 97331, USA
2 Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA
Reprint requests to: Dr. Max Deinzer, Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA; e-mail: max.deinzer{at}orst.edu; fax: (541) 737-0497.
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.
Keywords: Thioredoxin; heat denaturation; modified protein; hydrogen deuterium exchange; electrospray ionization mass spectrometry
Abbreviations: AMBER, assisted model building with energy refinement • CD, circular dichroism • CEC, S-(2-chloroethyl)cystein • CEG, S-(2-chloroethyl)glutathione • CID, collision-induced dissociation • F, folded state • H/D, hydrogen/deuterium • HPLC, high-performance liquid chromatography • MS, mass spectrometry • NMR, nuclear magnetic resonance • TCEP, Tris(2-carboxyethyl)phosphine • Tm, melting temperature • TRX, Escherichia coli thioredoxin • Oxi-, oxidized- • Red-, reduced- • GS-ethyl-, Cys-32-ethylglutathionylated- • Cys-ethyl-, Cys-32-ethylcysteinylated- • U, unfolded state • UV, ultraviolet
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