Intramolecular interactions in chemically modified Escherichia coli thioredoxin monitored by hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Site specific amide hydrogen/deuterium content of oxidized and reduced Esch erichia coli thioredoxin, and alkylated derivatives, Cys-32-ethylglutathion ylated and Cys-32-ethylcysteinylated thioredoxins are measured, after expos ure for 20 s to D2O/phosphate buffer (pH 5.7), by electrospray mass spectro metry. The degree of deuteration of Oxi-TRX and Red-TRX correlated with the rates of H/D exchange measured previously by NMR. The ethylcysteinyl modif ication was shown to minimally perturb the active site of the reduced prote in, but showed more global effects on structures of alpha -helices and beta -strands distant from the site of modification. In contrast, the larger et hylglutathionyl group had little effect on the protein's overall conformati on, but significantly affected the structure of loops close to the active s ite. A molecular model of GS-ethyl-TRX derived from molecular simulation al lowed the H/D exchange results to be interpreted in terms of specific inter actions between the alkyl chain and the protein surface. The specific confo rmation of the ethyl glutathione modification was predicted to be fixed by salt bridges between the carboxylates of the gamma -Glu and Gly of glutathi one and the guanidinium of Arg-73 and is an element of -amino group of Lys- 90 of the protein. Specific hydrogen bonding, interactions between the glut athione carbonyl oxygens and the amide protons of thioredoxin residues Ile- 75 and Ala-93 were predicted. The H/D exchange studies showed low levels of deuterium incorporation at backbone nitrogens of these residues. The data also provided evidence for an unusual amide proton-amide nitrogen hydrogen bond within the ethylglutathionylated chain. These same sets of electrostat ic and hydrogen bonding interactions were not predicted or observed for the smaller alkyl modification in Cys-ethyl-TRX.