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

dc.contributor.authorWelegedara, Adarshi Pen_AU
dc.contributor.authorAdams, Luke Aen_AU
dc.contributor.authorGraham, Bimen_AU
dc.contributor.authorOtting, Gottfrieden_AU
dc.contributor.authorHuber, Thomasen_AU
dc.date.accessioned2018-10-18T02:16:51Z
dc.date.issued2018-07-18
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.en_AU
dc.description.sponsorshipFinancial support by the Australian Research Council, including a Laureate Fellowship to G.O., is gratefully acknowledged.en_AU
dc.format.mimetypeapplication/pdfen_AU
dc.identifier.issn1043-1802en_AU
dc.identifier.urihttp://hdl.handle.net/1885/148490
dc.publisherAmerican Chemical Societyen_AU
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).en_AU
dc.sourceBioconjugate chemistryen_AU
dc.titleSite-Specific Incorporation of Selenocysteine by Genetic Encoding as a Photocaged Unnatural Amino Aciden_AU
dc.typeJournal articleen_AU
dcterms.accessRightsOpen Accessen_AU
local.bibliographicCitation.issue7en_AU
local.bibliographicCitation.lastpage2264en_AU
local.bibliographicCitation.startpage2257en_AU
local.contributor.affiliationOtting, G., Research School of Chemistry, The Australian National Universityen_AU
local.contributor.authoremailgottfried.otting@anu.edu.auen_AU
local.contributor.authoruidu4046684en_AU
local.identifier.citationvolume29en_AU
local.identifier.doi10.1021/acs.bioconjchem.8b00254en_AU
local.identifier.essn1520-4812en_AU
local.identifier.uidSubmittedByu1005913en_AU
local.publisher.urlhttps://pubs.acs.org/en_AU
local.type.statusAccepted Versionen_AU

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