Site-specific amide hydrogen/deuterium exchange in E. coli thioredoxins measured by electrospray ionization mass spectrometry.
Mass spectrometry as an analytical tool to study protein folding and structure by hydrogen/deuterium exchange is a relatively new approach. In this study, site-specific amide deuterium content was measured in oxidized and reduced E. coli thioredoxins by using the b(n) ions in electrospray ionization CID MS/MS experiments after 20-s incubation in D(2)O phosphate-buffered solution (pH 5.7). The deuterium levels correlated well with reported NMR-determined H/D exchange rate constants. The deuterium measured by y(n) ions, however, showed much less reliable correlation with rate exchange data. In general, residues in alpha helices and beta sheets, when measured by b(n) ions, showed low incorporation of deuterium while loops and turns had high deuterium levels. Most amide sites in the two protein forms showed similar deuterium levels consistent with the expected similarity of their structures, but there were some differences. The turn consisting of residues 18-22 in particular showed more variability in deuterium content consistent with reported structural differences in the two forms. The deuterium uptake by thioredoxins alkylated at Cys-32 by S-(2-chloroethyl)glutathione and S-(2-chloroethyl)cysteine, in peptides 1-24 and 45-58, was similar to that observed for oxidized and reduced thioredoxins, but several residues, particularly Leu-53 and Thr-54, showed slightly elevated deuterium levels, suggesting that structural changes had occurred from alkylation of the protein at Cys-32. It is concluded that b(n) ions are reliable for determining the extent of site-specific amide hydrogen isotope exchange and that mass spectrometry is useful as a complementary technique to NMR and other analytical methods for probing regional structural characteristics of proteins.
Kim
M Y
MY
Department of Chemistry, Oregon State University, Corvallis, OR 97331, USA.
Maier
C S
CS
Reed
D J
DJ
Deinzer
M L
ML
D000595:Amino Acid Sequence; D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism
D013879:Thioredoxins; D006859:Hydrogen; D003903:Deuterium