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

Adarshi P. Welegedara, Luke A. Adams, Thomas Huber, Bim Graham, Gottfried Otting

Research output: Contribution to journalArticleResearchpeer-review

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 tRNACUA. 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.

Original languageEnglish
Pages (from-to)2257-2264
Number of pages8
JournalBioconjugate Chemistry
Volume29
Issue number7
DOIs
Publication statusPublished - 18 Jul 2018

Cite this

Welegedara, Adarshi P. ; Adams, Luke A. ; Huber, Thomas ; Graham, Bim ; Otting, Gottfried. / Site-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid. In: Bioconjugate Chemistry. 2018 ; Vol. 29, No. 7. pp. 2257-2264.
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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 tRNACUA. 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.",
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Site-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid. / Welegedara, Adarshi P.; Adams, Luke A.; Huber, Thomas; Graham, Bim; Otting, Gottfried.

In: Bioconjugate Chemistry, Vol. 29, No. 7, 18.07.2018, p. 2257-2264.

Research output: Contribution to journalArticleResearchpeer-review

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