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Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry
Author(s) -
Kim MooYoung,
Maier Claudia S.,
Reed Donald J.,
Deinzer Max L.
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.3140102
Subject(s) - mass spectrometry , escherichia coli , chemistry , electrospray ionization , thioredoxin , electrospray , chromatography , analytical chemistry (journal) , biochemistry , enzyme , gene
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.