E. coli thioredoxin (TRX) was modified by the episulfonium ion derived
from S-(2-chloroethyl)glutathione (CEG) or S-(2-chloroethyl)cysteine (CEC). The
alkylation site was located at Cys-32, which was confirmed by tandem mass
spectrometry. Two forms of native TRX, Oxi- and Red-TRX, and two modified
TRXs, GS- and Cys-TRX, were examined by hydrogen/deuterium (H/D)
exchange reactions using electrospray ionization mass spectrometry (ESI-MS)
for the analysis.
Conformational dynamics during thermal denaturation were probed by H/D
exchange-in experiments. Under conditions in which the folded conformational
state is marginally stable, H/D exchange-in experiments resulted in mass spectra
differing in the number of incorporated deuteriums which indicates the presence
of two distinct populations of molecules. As the exchange-in time increased, the
population representing the unfolded state increased and the population for the
folded state decreased. The rate of conversion was used to estimate the rate
constant of unfolding. ESI mass spectra were also recorded as a function of
temperature without H/D exchange, and the observed bimodal charge state
distributions were analyzed in order to estimate melting temperatures. GS-TRX
showed increased resistance to hydrogen isotope exchange in comparison with
Red-TRX indicating that there were enhanced intramolecular interactions in the
former protein.
Pepsin digestion was performed on deuterated TRXs to analyze different
structural regions. The amount of deuterium incorporated was monitored with
peptic peptides from deuterated TRXs with different exchange-in incubation
periods. Deuterium levels of each peptide were plotted versus the exchange time
and fitted with a series of first-order rate terms. The regions 59-80 and 81-108 of
Oxi- and Red-TRX showed an EX1 mechanism as evidenced by two distinct
mass envelopes that appeared after a short incubation time in deuterated
solvent.
Tandem mass spectrometry (MS/MS) was carried out to obtain the
information on individual amide linkages. MS/MS data showed generally
excellent correlations with the exchange rate constants from published NMR data
on Oxi- and Red-TRXs. Two residues, Ile-75 and Ala-93 in GS-TRX indicated the
most probable sites responsible for induced H-bonding by the attached
glutathionyl group, which was consistent with the energy minimized structure
predicted by AMBER force field constants. / Graduation date: 2001
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/37224 |
| Date | 13 December 2000 |
| Creators | Kim, Moo-young |
| Contributors | Deinzer, Max L. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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