Identifying cysteine residues susceptible to oxidation by photoactivatable atomic oxygen precursors using a proteome-wide analysis.

The reactivity profile of atomic oxygen [O(3P)] in the condensed phase has shown a preference for the thiol group of cysteines. In this work, water-soluble O(3P)-precursors were synthesized by adding aromatic burdens and water-soluble sulphonic acid groups to the core structure of dibenzothiophene-S-oxide (DBTO) to study O(3P) reactivity in cell lysates and live cells. The photodeoxygenation of these compounds was investigated using common intermediates, which revealed that an increase in aromatic burdens to the DBTO core structure decreases the total oxidation yield due to competitive photodeoxygenation mechanisms. These derivatives were then tested in cell lysates and live cells to profile changes in cysteine reactivity using the isoTOP-ABPP chemoproteomics platform. The results from this analysis indicated that O(3P) significantly affects cysteine reactivity in the cell. Additionally, O(3P) was found to oxidize cysteines within peptide sequences with leucine and serine conserved at the sites surrounding the oxidized cysteine. O(3P) was also found to least likely oxidize cysteines among membrane proteins.

Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine.

Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.