Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNA CUA pair that directs the efficient incorporation of phosphoserine into recombinant proteins in E. coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a non-hydrolyzable analog of phosphoserine. Our approach enables quantitative decoding of the amber stop codon as phosphoserine and we purify several milligrams-per-liter of proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access: including phosphorylated ubiquitin and a kinase (Nek7) that is synthetically activated by a genetically encoded phosphorylation in its activation loop.The phosphorylation of proteins in eukaryotic cells is a key post-translational modification that regulates essential and diverse biological phenomena. Phosphorylation is installed by protein kinases on serine, threonine, tyrosine and histidine residues, and removed by phosphatases, and serine phosphorylation is the most abundant phosphorylation observed in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.

Deoxyribozymes (DNA catalysts) have been reported for cleavage of RNA phosphodiester linkages, but cleaving peptide or DNA phosphodiester linkages is much more challenging. Using in vitro selection, here we identified deoxyribozymes that sequence-specifically hydrolyze DNA with multiple turnover and rate enhancement of 108 (possibly as high as 1014). The new DNA catalysts require both Mn2+ and Zn2+, which is intriguing because many natural DNA nucleases are bimetallic protein enzymes.

Identifying the proteins synthesized in defined cells at specific times in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.Defining the proteins synthesized in specific cells, at specific times within animals will provide molecular insight into diverse biological processes including development, 1 differentiation, 1 circadian oscillations, 2 morphogenesis 3 and learning and memory. 4 Despite the importance of this challenge, there are no general methods for identifying the proteins synthesized in specific cells at specific times within an animal.

Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.

Fluorophores have transformed the way we study biological systems, enabling noninvasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids (FlAAs) have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein-protein interactions in situ or imaging nanoscopic events in real-time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of FlAAs and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using sitespecific genetic encoding and bio-orthogonal approaches, and their application to design novel artificial proteins as well as to investigate biological processes in cells by means of optical imaging.

We show that DNA catalysts (deoxyribozymes, DNA enzymes) can phosphorylate tyrosine residues of peptides. Using in vitro selection, we identified deoxyribozymes that transfer the γ-phosphoryl group from a 5′-triphosphorylated donor (a pppRNA oligonucleotide or GTP) to the tyrosine hydroxyl acceptor of a tethered hexapeptide. Tyrosine kinase deoxyribozymes that use pppRNA were identified from each of N30, N40, and N50 random-sequence pools. Each deoxyribozyme requires Zn2+, and most additionally require Mn2+. The deoxyribozymes have little or no selectivity for the amino acid identities near the tyrosine, but they are highly selective for phosphorylating tyrosine rather than serine. Analogous GTP-dependent DNA catalysts were identified and found to have apparent Km(GTP) as low as ca. 20 μM. These findings establish that DNA has the fundamental catalytic ability to phosphorylate the tyrosine side chain of a peptide substrate.

New deoxyribozymes are shown to catalyze reactions of serine ide chains, forming nucleopeptide linkages and discriminating etween serine and tyrosine or between two competing serines.Since the identification of the first artificial deoxyribozyme, which catalyzes RNA phosphodiester bond cleavage, 1 many DNA enzymes have been found to catalyze reactions that typically involve oligonucleotide substrates. 2-4 A major challenge is to incorporate small molecules, proteins, and other substrates into the repertoire of DNA catalysis. Towards this goal, we recently reported the Tyr1 deoxyribozyme, which catalyzes the nucleophilic attack of a tyrosine phenolic OH group into 5′-triphosphate-RNA, leading to formation of a nucleopeptide linkage. 5 Covalent linkages between nucleic acids and proteins are integral to the mechanisms of topoisomerases 6 and recombinases 7 and are found in many other biological contexts. 8 The Tyr1 deoxyribozyme was identified by in vitro selection, which iteratively searches through ~10 14 random DNA sequences to find those with catalytic activity. 9 During the Tyr1 selection process, tyrosine was presented at the intersection of a three-helix junction (3HJ) formed from candidate deoxyribozyme sequences and two nucleic acid strands. One of these strands comprised DNA containing the embedded tyrosine, whereas the other strand was 5′-triphosphate-RNA (Fig. 1a). The 3HJ architecture, inspired by the discovery of 7S11 and related deoxyribozymes that catalyze formation of 2′,5′-branched RNA, 10 spatially juxtaposes a nucleophile (e.g., tyrosine OH group) and electrophile (5′-triphosphate). This design allows interrogation of DNA sequences while focusing primarily on their intrinsic ability to catalyze the desired chemical reaction, without requiring that the DNA also bind a separate free substrate molecule.Alongside the successful identification of Tyr1, parallel selection experiments were previously performed in which a single serine residue (rather than tyrosine) was placed at the 3HJ intersection (Fig. 1a), but essentially no active deoxyribozymes were identified. 5 The serine aliphatic hydroxyl group is less reactive than the tyrosine phenolic hydroxyl group, providing a greater catalytic challenge. In the present report, for the first time we have achieved robust DNA-catalyzed reactivity of the serine side chain, by expanding the structural context from a single amino acid to an Ala-Ser-Ala tripeptide while retaining the 3HJ architecture (Fig. 1b) The new experiments used our previously established selection strategy 11 to identify catalytic DNA sequences. At the outset of each selection round, the 5′-triphosphate-RNA was ligated via its 3′-terminus to the 5′-end of the deoxyribozyme pool strand. Any DNA sequence that successfully joins the amino acid side chain of the tripeptide-containing substrate to the 5′-triphosphate-RNA as shown in Fig. 1 becomes separable from even a substantial excess of catalytically inactive DNA sequences by polyacrylamide gel electrophoresis (PAGE). During the...