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

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

Description

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...[Show more]

dc.contributor.authorWelegedara, Adarshi P
dc.contributor.authorAdams, Luke A
dc.contributor.authorGraham, Bim
dc.contributor.authorOtting, Gottfried
dc.contributor.authorHuber, Thomas
dc.date.accessioned2018-10-18T02:16:51Z
dc.identifier.issn1043-1802
dc.identifier.urihttp://hdl.handle.net/1885/148490
dc.description.abstractSelenocysteine (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.
dc.description.sponsorshipFinancial support by the Australian Research Council, including a Laureate Fellowship to G.O., is gratefully acknowledged.
dc.format.mimetypeapplication/pdf
dc.publisherAmerican Chemical Society
dc.rights© 2018 American Chemical Society. http://www.sherpa.ac.uk/romeo/issn/1043-1802/..."author can archive post-print (ie final draft post-refereeing) If mandated by funding agency or employer/ institution. 12 months embargo" from SHERPA/RoMEO site (as at 18/10/18).
dc.sourceBioconjugate chemistry
dc.titleSite-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Acid
dc.typeJournal article
local.identifier.citationvolume29
dc.date.issued2018-07-18
local.publisher.urlhttps://pubs.acs.org/
local.type.statusAccepted Version
local.contributor.affiliationOtting, G., Research School of Chemistry, The Australian National University
local.description.embargo2019-07-18
local.identifier.essn1520-4812
local.bibliographicCitation.issue7
local.bibliographicCitation.startpage2257
local.bibliographicCitation.lastpage2264
local.identifier.doi10.1021/acs.bioconjchem.8b00254
dcterms.accessRightsOpen Access
CollectionsANU Research Publications

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