No abstract

We demonstrate that an orthogonal Methanosarcina barkeri MS pyrrolysyl-tRNA synthetase/tRNA(CUA) pair directs the efficient, site-specific incorporation of N6-[(2-propynyloxy)carbonyl]-L-lysine, containing a carbon-carbon triple bond, and N6-[(2-azidoethoxy)carbonyl]-L-lysine, containing an azido group, into recombinant proteins in Escherichia coli. Proteins containing the alkyne functional group are labeled with an azido biotin and an azido fluorophore, via copper catalyzed [3+2] cycloaddition reactions, to produce the corresponding triazoles in good yield. The methods reported are useful for the site-specific labeling of recombinant proteins and may be combined with mutually orthogonal methods of introducing unnatural amino acids into proteins as well as with chemically orthogonal methods of protein labeling. This should allow the site specific incorporation of multiple distinct probes into proteins and the control of protein topology and structure by intramolecular orthogonal conjugation reactions.

Precise photochemical control of protein function can be achieved through the site-specific introduction of caging groups. Chemical and enzymatic methods, including in vitro translation and chemical ligation, have been used to photocage proteins in vitro. These methods have been extended to allow the introduction of caged proteins into cells by permeabilization or microinjection, but cellular delivery remains challenging. Since lysine residues are key determinants for nuclear localization sequences, the target of key post-translational modifications (including ubiquitination, methylation, and acetylation), and key residues in many important enzyme active sites, we were interested in photocaging lysine to control protein localization, post-translational modification, and enzymatic activity. Photochemical control of these important functions mediated by lysine residues in proteins has not previously been demonstrated in living cells. Here we synthesized 1 and evolved a pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode the incorporation of this amino acid in response to an amber codon in mammalian cells. To exemplify the utility of this amino acid, we caged the nuclear localization sequences (NLSs) of nucleoplasmin and the tumor suppressor p53 in human cells, thus mislocalizing the proteins in the cytosol. We triggered protein nuclear import with a pulse of light, allowing us to directly quantify the kinetics of nuclear import.