Genetically
introducing novel chemical bonds into proteins provides
innovative avenues for biochemical research, protein engineering,
and biotherapeutic applications. Recently, latent bioreactive unnatural
amino acids (Uaas) have been incorporated into proteins to covalently
target natural residues through proximity-enabled reactivity. Aryl
fluorosulfate is particularly attractive due to its exceptional biocompatibility
and multitargeting capability via sulfur(VI) fluoride exchange (SuFEx)
reaction. Thus far, fluorosulfate-l-tyrosine (FSY) is the
only aryl fluorosulfate-containing Uaa that has been genetically encoded.
FSY has a relatively rigid and short side chain, which restricts the
diversity of proteins targetable and the scope of applications. Here
we designed and genetically encoded a new latent bioreactive Uaa,
fluorosulfonyloxybenzoyl-l-lysine (FSK), in E. coli and mammalian cells. Due to its long and flexible aryl fluorosulfate-containing
side chain, FSK was particularly useful in covalently linking protein
sites that are unreachable with FSY, both intra- and intermolecularly, in vitro and in live cells. In addition, we created covalent
nanobodies that irreversibly bound to epidermal growth factor receptors
(EGFR) on cells, with FSK and FSY targeting distinct positions on
EGFR to counter potential mutational resistance. Moreover, we established
the use of FSK and FSY for genetically encoded chemical cross-linking
to capture elusive enzyme–substrate interactions in live cells,
allowing us to target residues aside from Cys and to cross-link at
the binding periphery. FSK complements FSY to expand target diversity
and versatility. Together, they provide a powerful, genetically encoded,
latent bioreactive SuFEx system for creating covalent bonds in diverse
proteins in vitro and in vivo, which
will be widely useful for biological research and applications.
Post‐translational modifications regulate protein structure and function. Lysine benzoylation is a newly discovered histone modification with unique physiological relevance. To construct proteins with this modification site‐specifically, we generated orthogonal tRNAPyl‐MaBzKRS pairs by engineering Methanomethylophilus alvus pyrrolysyl‐tRNA synthetase, allowing the genetic incorporation of ϵ‐N‐benzoyllysine (BzK) into proteins with high efficiency in E. coli and mammalian cells. Two types of MaBzKRS were identified to incorporate BzK using mutations located at different positions of the amino acid binding pocket. These MaBzKRS are small in size and highly expressed, which will afford broad utilities in studying the biological effects of lysine benzoylation.
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