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
10.1021/acs.bioconjchem.8b00254.s001
https://acs.figshare.com/articles/journal_contribution/Site-Specific_Incorporation_of_Selenocysteine_by_Genetic_Encoding_as_a_Photocaged_Unnatural_Amino_Acid/6635534
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, <i>Mm</i> PCC2RS, and its
cognate tRNA<sub>CUA</sub>. 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.
2018-06-06 00:00:00
Methanosarcina mazei pyrrolysyl-tRNA synthetase
Mm PCC 2RS
Sec incorporation
site-specific covalent protein modification
acid
site-specific protein modifications
SECIS
UV
study ligand binding
selenocysteine insertion sequence
CUA
Photocaged Unnatural Amino
NMR
solvent-exposed cysteine residues