A Genetically Encoded Photocaged Tyrosine

Deiters, Alexander; Groff, Dan; Ryu, Yonguk; Xie, Jianming; Schultz, Peter G.

doi:10.1002/anie.200600264

Cited by 188 publications

(133 citation statements)

References 20 publications

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“…They started the evolution of the orthogonal pair from a mutant of the Methanocaldococcus jannaschii tyrosyl-tRNA synthetase for the incorporation of o-nitrobenzyltyrosine. 94 When the three analogs were incorporated into the chromophore (Tyr66), the 2-fluoro and the 2,6-difluoro derivative exhibited a blue shift in the emission profiles of the superfolder GFP (503 and 509 nm compared to 513 of the parent protein). In contrast, fluorine in position 3 leads to a small red shift (515 nm).…”

Section: The Stop Codon Suppression (Scs) Methodologymentioning

confidence: 99%

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Merkel

Budiša

2012

Org. Biomol. Chem.

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Section: The Stop Codon Suppression (Scs) Methodologymentioning

confidence: 99%

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Merkel

Budiša

2012

Org. Biomol. Chem.

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“…More recently, photochemically reactive amino acids have been used as probes of protein function in vitro and in cells. For example, photocaged cysteine (13), serine (14), lysine (15), and tyrosine (16) derivatives have been site-specifically incorporated into proteins to allow the photodynamic regulation of their activity (15,(37)(38)(39). In one example, the localization of the yeast transcription factor Pho4 was followed in living cells by selectively blocking its phosphorylation with a photocaged serine.…”

Section: Expanded Genetic Codementioning

confidence: 99%

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Young

Schultz

2010

Journal of Biological Chemistry

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306

237

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The ability to genetically encode unnatural amino acids beyond the common 20 has allowed unprecedented control over the chemical structures of recombinantly expressed proteins. Orthogonal aminoacyl-tRNA synthetase/tRNA pairs have been used together with nonsense, rare, or 4-bp codons to incorporate >50 unnatural amino acids into proteins in Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, and mammalian cell lines. This has allowed the expression of proteins containing amino acids with novel side chains, including fluorophores, post-translational modifications, metal ion chelators, photocaged and photocross-linking moieties, uniquely reactive functional groups, and NMR, IR, and x-ray crystallographic probes. Early MethodologyWith few exceptions, all organisms are restricted to the 20 common amino acid building blocks for the ribosomal biosynthesis of proteins. However, it is clear that proteins require a higher level of chemical complexity for many functions, as evidenced by the frequent use of post-translational modifications and the dependence of many enzymes on cofactors. Thus, the addition of new amino acid building blocks to the genetic code should further expand the range of functions available to proteins and provide powerful new tools for probing protein structure and function both inside and outside of living cells (1). Although recent advances in synthetic and semisynthetic methods have proven very useful for the incorporation of unnatural amino acids into proteins, they are generally limited by low yields and are technically cumbersome in the production of larger proteins. The use of the cellular biosynthetic machinery to introduce novel amino acids abrogates issues relating to scalability and protein size and simplifies the study of modified proteins in living cells.To add a new amino acid to the genetic repertoire, a codon is needed that uniquely specifies that amino acid. The 20 canonical amino acids are encoded by 61 degenerate triplet codons, leaving the remaining three codons (TAG, amber; TAA, ochre; and TGA, opal) to serve as translational stops. Previously, we and others (2) used the redundancy of these "blank" stop codons, together with the ribosomal machinery, to site-specifically incorporate unnatural amino acids into proteins in vitro in response to the amber nonsense codon. This was accomplished by chemically aminoacylating a nonsense suppressor tRNA (tRNA CUA , the amber suppressor tRNA) with the desired unnatural amino acid and adding the aminoacyl-tRNA to a cell-free transcription/translation system along with the gene of interest harboring a TAG mutation at the target site. In the decade that followed, this method was used to site-specifically incorporate a large number of unnatural amino acids with a wide variety of structures into proteins (3, 4). These amino acids were used to probe the roles of specific amino acid side chains and backbone groups in protein folding and stability, catalytic mechanisms, and biomolecular interactions. Later, this approach was extended to th...

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“…[4][5][6][7][8][9][10] For the generation of caged proteins in live cells, the genetic code can be expanded allowing for the site-specific incorporation of caged amino acids into proteins. [11][12][13][14][15][16][17][18][19][20] The caged amino acids are inserted in response to an amber stop codon, TAG, by suppressor tRNAs that have been misacylated by engineered tRNA synthetases. [21][22][23][24][25][26][27][28] Advantages of this methodology over other approaches are that the location of the unnatural amino acid is determined through genetic mutagenesis and the light-activated proteins are produced in live cells eliminating the requirement for transfection or injection.…”

Section: Introductionmentioning

confidence: 99%

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

et al. 2013

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Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the sitespecific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.

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A Genetically Encoded Photocaged Tyrosine

Cited by 188 publications

References 20 publications

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

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