Photocleavage of the Polypeptide Backbone by 2-Nitrophenylalanine

Peters, Francis B.; Brock, Ansgar; Wang, Jiangyun; Schultz, Peter G.

doi:10.1016/j.chembiol.2009.01.013

Cited by 60 publications

(70 citation statements)

References 21 publications

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“…For example, this approach has been used to cross-link membrane protein ligands, such as G protein-coupled receptor peptide ligands, in S. cerevisiae (42). Finally, to selectively cleave proteins with light, o-nitrophenylalanine (18) has been genetically encoded, which results in the site-specific scission of the protein backbone through a light-induced radical mechanism (43).…”

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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313

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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Section: Expanded Genetic Codementioning

confidence: 99%

Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon

Young

Schultz

2010

Journal of Biological Chemistry

Self Cite

313

237

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“…Our main rationale of using this photolabile group was because of its commercial availability, as well as its known photolysis mechanisms [34]. Peters et al have shown that photolysis of acetylated peptides containing 2-NPA leads to the cleavage of peptide backbone and produces a C-terminal carboxylate group and an N-terminal cinnoline group (Scheme 1C) [34]. It is worth noting that other photo-sensitive motifs (e.g., o -nitrobenzyl groups) could also be used to achieve UV-mediated photocleavage.…”

Section: Resultsmentioning

confidence: 99%

Facile preparation of photodegradable hydrogels by photopolymerization

Han

Lin

2013

Polymer

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Photodegradable hydrogels have emerged as a powerful material platform for studying and directing cell behaviors, as well as for delivering drugs. The premise of this technique is to use a cytocompatible light source to cleave linkers within a hydrogel, thus causing reduction of matrix stiffness or liberation of matrix-tethered biomolecules in a spatial-temporally controlled manner. The most commonly used photodegradable units are molecules containing nitrobenzyl moieties that absorb light in the ultraviolet (UV) to lower visible wavelengths (~280 to 450 nm). Because photodegradable linkers and hydrogels reported in the literature thus far are all sensitive to UV light, highly efficient UV-mediated photopolymerizations are less likely to be used as the method to prepare these hydrogels. As a result, currently available photodegradable hydrogels are formed by redox-mediated radical polymerizations, emulsion polymerizations, Michael-type addition reactions, or orthogonal click chemistries. Here, we report the first photodegradable poly(ethylene glycol)-based hydrogel system prepared by step-growth photopolymerization. The model photolabile peptide cross-linkers, synthesized by conventional solid phase peptide synthesis, contained terminal cysteines for step-growth thiol-ene photo-click reactions and a UV-sensitive 2-nitrophenylalanine residue in the peptide backbone for photo-cleavage. Photolysis of this peptide was achieved through adjusting UV light exposure time and intensity. Photopolymerization of photodegradable hydrogels containing photolabile peptide cross-linkers was made possible via a highly efficient visible light-mediated thiol-ene photo-click reaction using a non-cleavage type photoinitiator eosin-Y. Rapid gelation was confirmed by in situ photo-rheometry. Flood UV irradiation at controlled wavelength and intensity was used to demonstrate the photodegradability of these photopolymerized hydrogels.

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“…To date, a wide range of chemical functionalities have been introduced into proteins via unnatural amino acids including, azides, alkynes, fluorophores, photosensitive moieties, and metal binders to name a few. [4][5][6][7][8][9][10][11] Specifically, modifying proteins with fluorescent probes provides the ability to examine structure and function of proteins as well as visualize their cellular location via fluorescence spectroscopy. 12 Numerous fluorescent probes have been utilized within a protein context to further advance our understanding of proteins.…”

Section: Introductionmentioning

confidence: 99%