Detection of Selenium/Sulphur substitution in the heterologous expression of Thioredoxin isoform 1 (Trx1) fromSaccharomyces cerevisiae

Abstract

Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous translation. Furthermore, the Maldi-TOF spectra are showing the presence of the most abundant isotopes of selenium inserted in the peptides containing cysteine and methionine, derived from the Trx1 digestion. The one dimensional 77Se–1H heteronuclear multiple quantum coherence NMR spectroscopy (1D-HMQC) for reduced Seleno substituted Trx1 (Se-Trx1), revealed three ressonance lines for 1Hβ1 from Selenocysteines 30 and 33, between 1.6 and 2,0 ppm. The bidimensional HMQC spectra (2D-HMQC) of the reduced Se-Trx1 show the 77 Se ressonance signal in 178 ppm, coupled with 1Hβ1 and 1Hβ2 lines between 2.1 and 1.8 ppm. The 1D-HMQC for oxidized Trx1 revealed the only one broad resonance in 2.6 ppm probably relative to the 1Hβ1 prótons. The 2D-HMQC spectrum of oxidized protein shows a higher chemical shift of selenocysteine 77Se (832 ppm) if compared to reduced state (178 ppm). Together these data are showing that the protocol of Se – S substitution developed here is a efficient method to label the active site of Thioredoxin 1 with a broad band chemical shift atom 77Se. Furthermore the large spectral window of the 77Se NMR detected between reduced and oxidized states of the Thioredoxin 1 shows that this atom is an excellent probe for accessing oxidative states and probably the conformational dynamics of the active site of the Se-Trx1.