Site-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid

Abstract

Selenocysteine (Sec) is a naturally occurring amino acid that is also referred to as the 21st amino acid. Site-specific incorporation of Sec into proteins is attractive, because the reactivity of a selenol group exceeds that of a thiol group and thus allows site-specific protein modifications. It is incorporated into proteins by an unusual enzymatic mechanism which, in E. coli and other organisms, involves the recognition of a selenocysteine insertion sequence (SECIS) in the mRNA of the target protein. Reengineering of the natural machinery for Sec incorporation at arbitrary sites independent of SECIS elements, however, is challenging. Here we demonstrate an alternative route, whereby a photocaged selenocysteine (PSc) is incorporated as an unnatural amino acid in response to an amber stop codon, using a mutant Methanosarcina mazei pyrrolysyl-tRNA synthetase, Mm PCC2RS, and its cognate tRNA. Following decaging by UV irradiation, proteins synthesized with PSc are readily tagged, e.g., with NMR probes to study ligand binding by NMR spectroscopy. The approach provides a facile route for genetically encoded Sec incorporation. It allows the production of pure selenoproteins and the Sec residue enables site-specific covalent protein modification with reagents that would usually react first with naturally occurring cysteine residues. The much greater reactivity of Sec residues allows their selective alkylation in the presence of highly solvent-exposed cysteine residues.

Trimethylsilyl reporter groups for NMR studies of conformational changes in G protein‐coupled receptors

“…Becker et al used chemical shifts of TMS tags to monitor ligand‐binding events of two different drug targets, namely, the Zika virus NS2B–NS3 protease and the human prolyl isomerase FK506 binding protein, thereby demonstrating that this tag is feasible for probing the interaction between soluble proteins and ligands by NMR. Welegedara et al used TMS tags in site‐specific alkylation of selenoproteins followed by studying ligand binding by 1 H NMR spectroscopy. All of these studies made use of a one‐step process that resulted in a stable thioether bond.…”

Real-time nuclear magnetic resonance spectroscopy in the study of biomolecular kinetics and dynamics

“…Most importantly, the approach relies on the presence of a chromophore within the NMR sample. This can either be a biomolecule carrying a photoactive group such as the yellow protein (Derix et al, 2003) or folding can be initiated by release of cofactors from photo-labile chelators (Kühn and Schwalbe, 2000), from caged ligands (Buck et al, 2007) or from photo-labile precursors that cage biomolecular conformation (Wenter et al, 2005).…”

Real-time NMR spectroscopy in the study of biomolecular kinetics and dynamics

“…Here, we will focus on the photo-caged amino acids, represented e.g. by o-nritobenzyl (o-NB) caged tyrosine (Deiters et al, 2006), cysteine (Wu et al, 2004), lysine (Chen et al, 2009), the 4,5-dimethoxy-2-nitrobenzyl caged serine (DMNB) (Lemke et al, 2007) and 1-Bromo-1-[4′,5′-(methylenedioxy)-2′-nitrophenyl]ethane caged selenocysteine (Welegedara et al, 2018). These caging groups have different photophysical properties: o-NB and the selenocysteine are cleaved by UV illumination and DMNB by blue visible light.…”

“…Photo-caged amino acids can also be used to allow for selective covalent modifications in proteins after cleavage of the cage. With site specific incorporation of a photo-caged selenocysteine and following uncaging, it is possible to site-specific modify these due to their higher reactivity in comparison to competing cysteine residues (Welegedara et al, 2018).…”