Histidine is a unique amino acid with an imidazole side chain in which both of the nitrogen atoms are capable of serving as a proton donor and proton acceptor in hydrogen bonding interactions. In order to probe the functional role of histidine involved in hydrogen bonding networks, fine-tuning the hydrogen bonding potential of the imidazole side chain is required but not feasible through traditional mutagenesis methods. Here, we show that two close mimetics of histidine, 3-methyl-histidine and thiazole alanine, can be genetically encoded using engineered pyrrolysine incorporation machinery. Replacement of the three histidine residues predicted to be involved in an extended charge-relay system in alanine racemase with 3-methyl-histidine or thiazole alanine shows a dramatic loss in the enzyme’s catalytic efficiency, implying the role of this extended charge-relay system in activating the active site residue Y265, a general acid/base catalyst in the enzyme.

As a member of a highly conserved family of NAD+-dependent histone deacetylases, Sirt6 is a key regulator of mammalian genome stability, metabolism, and life span. Previous studies indicated that Sirt6 is hardwired to remove histone acetylation at H3K9 and H3K56. However, how Sirt6 recognizes its nucleosome substrates has been elusive due to the difficulty of accessing homogeneous acetyl-nucleosomes and the low activity of Sirt6 toward peptide substrates. Based on the fact that Sirt6 has an enhanced activity to remove long chain fatty acylation from lysine, we developed an approach to recombinantly synthesize histone H3 with a fatty acylated lysine, Nε-(7-octenoyl)-lysine (OcK), installed at a number of lysine sites and used these acyl-H3 proteins to assemble acyl-nucleosomes as active Sirt6 substrates. A chemical biology approach that visualizes OcK in nucleosomes and therefore allows direct sensitization of Sirt6 activities on its acyl-nucleosome substrates was also formulated. By combining these two approaches, we showed that Sirt6 actively removes acylation from H3K9, H3K18, and H3K27; has relatively low activities toward H3K4 and K3K23; but sluggishly removes acylation at H3K14, H3K36, H3K56, and H3K79. Overexpressing Sirt6 in 293T cells led to downregulated acetylation at H3K18 and K3K27, confirming these two novel Sirt6-targeted nucleosome lysine sites in cells. Given that downregulation of H3K18 acetylation is correlated with a poor prognosis of several cancer types and H3K27 acetylation antagonizes repressive gene regulation by di- and trimethylation at H3K27, our current study implies that Sirt6 may serve as a target for cancer intervention and regulatory pathway investigation in cells.

Lysine acetylation serves as an epigenetic marker for myriad cellular processes, such as signaling, differentiation, DNA repair, angiogenesis, and the like. Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent histone deacetylases that operate as post-translational regulators for the deacetylation of acetyllysine. Here, we discuss the ability for SIRT1 and SIRT2 to deacetylate monoacetylated histone H3 on two separate architectures—the peptide and the nucleosome. In addition, we analyze the site-specificity of SIRT1 and SIRT2 on 10 different monoacetylated histone H3 nucleosomes. By utilizing a rapid screening array, SIRT1 and SIRT2 were found to demonstrate heightened enzymatic activity when incubated with nucleosomal substrates over their peptide counterparts. These two enzymes displayed little site-specificity among the acetyl-nucleosomes screened, contrary to previous expectations, as well. The implication of the overall nonspecificity of SIRT1 and SIRT2 on the nucleosome suggests that these sirtuin enzymes have an adaptive nature, harnessing an ability to respond to various cellular situations, rather than an enzyme specifically designed for a particular task or function.

The biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products typically involves a precursor peptide which contains a leader peptide that is important for the modification process, and that is removed in the final step by a protease. Genome mining efforts for new RiPPs are often hampered by the lack of a general method to remove the leader peptides. We describe here the incorporation of hydroxy acids into the precursor peptides in E. coli which results in connection of the leader peptide via an ester linkage that is readily cleaved by simple hydrolysis. We demonstrate the method for two lantibiotics, lacticin 481 and nukacin ISK-1.

Controlled orientation of a small laccase on a multi-walled carbon nanotube electrode was achieved via copper-free click chemistry mediated immobilization. Modification of the enzyme was limited to only the tethering site and involved the genetic incorporation of the unnatural amino acid 4-azido-L-phenylalanine (AzF). This approach enabled efficient direct electron transfer.

Using the amber suppression approach, four noncanonical amino acids (ncAAs) were used to replace existing amino acids at four positions in lasso peptide microcin J25 (MccJ25). The lasso peptide biosynthesis enzymes tolerated all four ncAAs and produced antibiotics with efficacy equivalent to wild-type in some cases. Given the rapid expansion of the genetically encoded ncAA pool, this study is the first to demonstrate an expedient method to significantly increase the chemical diversity of lasso peptides.

Correction for ‘Genetically encoded unstrained olefins for live cell labeling with tetrazine dyes’ by Yan-Jiun Lee et al., Chem. Commun., 2014, DOI: 10.1039/c4cc06435f.

A number of non-canonical amino acids (NCAAs) with unstrained olefins are genetically encoded using mutant pyrrolysyl-tRNA synthetase–tRNAPylCUA pairs. These NCAAs readily undergo inverse electron-demand Diels–Alder cycloadditions with tetrazine dyes, leading to selective labeling of proteins bearing these NCAAs in live cells.

The transient formation of nitrilimine in aqueous conditions is greatly influenced by pH and chloride. In basic conditions (pH 10) with no chloride, a diarylnitrilimine precursor readily ionizes to form diarylnitrilimine that reacts almost instantly with an acrylamide-containing protein and fluorescently labels it.

Thirteen novel non-canonical amino acids were synthesized and tested for suppression of an amber codon using a mutant pyrrolysyl-tRNA synthetase–tRNAPylCUA pair. Suppression was observed with varied efficiencies. One non-canonical amino acid in particular contains an azide that can be applied for site-selective protein labeling.

Using a mutant pyrrolysyl-tRNA synthetase-tRNAPylCUA pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling.

Detailed kinetic analyses of inverse electron-demand Diels–Alder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these “click” reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNACUAPyl pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.

Seven phenylalanine derivatives with small ortho substitutions were genetically encoded in Escherichia coli and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNACUAPyl. Ortho substitutions of the phenylalanine derivatives reported herein include three halides, methyl, methoxy, nitro, and nitrile. These compounds have the potential for use in multiple biochemical and biophysical applications. Specifically, we demonstrated that o-cyano-phenylalanine could be used as a selective sensor to probe the local environment of proteins and applied this to study protein folding/unfolding. For six of these compounds this constitutes the first report of their genetic incorporation in living cells. With these compounds the total number of substrates available for PylRS(N346A/C348A) is increased to nearly 40, which demonstrates that PylRS(N346A/C348A) is able to recognize phenylalanine with a substitution at any side-chain aromatic position as a substrate. To our knowledge, PylRS(N346A/C348A) is the only aminoacyl-tRNA synthetase with such a high substrate promiscuity.

Nε-Acryloyl-l-lysine, a noncanonical amino acid with an electron deficient olefin, is genetically encoded in Escherichia coli using a pyrrolysyl-tRNA synthetase mutant in coordination with tRNACUAPyl. The acrylamide moiety is stable in cells, whereas it is active enough to perform a diverse set of unique reactions for protein modifications in vitro. These reactions include 1,4-addition, radical polymerization, and 1,3-dipolar cycloaddition. We demonstrate that a protein incorporated with Nε-acryloyl-l-lysine is efficiently modified with thiol-containing nucleophiles at slightly alkali conditions, and the acrylamide moiety also allows rapid radical copolymerization of the same protein into a polyacrylamide hydrogel at physiological pH. At physiological conditions, the acrylamide functionality undergoes a fast 1,3-dipolar cycloaddition reaction with diaryl nitrile imine to show turn-on fluorescence. We have used this observation to demonstrate site-specific fluorescent labeling of proteins incorporated with Nε-acryloyl-l-lysine both in vitro and in living cells. This critical development allows easy access to an array of modified proteins for applications where high specificity and reaction efficiency are needed.

When coexpressed with its cognate amber suppressing tRNACUAPyl, a pyrrolysyl-tRNA synthetase mutant N346A/C348A is able to genetically incorporate 12 meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro, ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone, alkyne, and azide moieties can be applied to site-specifically label proteins with fluorescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.

Using an evolved pyrrolysyl-tRNA synthetase-tRNAPyl pair, a Se-alkylselenocysteine was genetically incorporated into histone H3 with a high protein expression yield. Quantitative oxidative elimination of Se-alkylselenocysteine followed by Michael addition reactions with various thiol nucleophiles generated biologically active mimics of H3 with posttranslational modifications including lysine methylation, lysine acetylation, and serine phosphorylation.

Using evolved pyrrolysyl-tRNA synthetase–tRNAPylCUA pairs, L-phenylalanine, p-iodo-L-phenylalanine and p-bromo-L-phenylalanine have been genetically incorporated into proteins at amber mutation sites in E. coli.

Posttranslational modifications modulate the activities of most eukaryotic proteins and play a critical role in all aspects of cellular life. Understanding functional roles of these modifications requires homogenously modified proteins that are usually difficult to purify from their natural sources. An emerging powerful tool for synthesis of proteins with defined posttranslational modifications is to genetically encode modified amino acids in living cells and incorporate them directly into proteins during the protein translation process. Using this approach, homogenous proteins with tyrosine sulfation, tyrosine phosphorylation mimics, tyrosine nitration, lysine acetylation, lysine methylation, and ubiquitination have been synthesized in large quantities. In this review, we provide a brief introduction to protein posttranslational modifications and the genetic noncanonical amino acid (NAA) incorporation technique, then discuss successful applications of the genetic NAA incorporation approach to produce proteins with defined modifications, and end with challenges and ongoing methodology developments for synthesis of proteins with other modifications.

A photocaged Nε-methyl-L-lysine has been genetically incorporated into proteins at amber codon positions in Escherichia coli using an evolved pyrrolysyl-tRNA synthetase-pylT pair. Its genetic incorporation and following photolysis to recover Nε-methyl-L-lysine at physiological pH provide a convenient method for the biosynthesis of proteins with monomethylated lysines at specific sites.

By overexpressing the C-terminal domain of the ribosomal protein L11 to decrease release factor 1-mediated termination of protein translation, enhanced amber suppression is achieved in E. coli. This enhanced amber suppression efficiency allows the genetic incorporation of three Nε-acetyl-L-lysines into one GFPUV protein in E. coli.

Over 300 amino acids are found in proteins in nature, yet typically only 20 are genetically encoded. Reassigning stop codons and use of quadruplet codons emerged as the main avenues for genetically encoding non-canonical amino acids (NCAAs). Canonical aminoacyl-tRNAs with near-cognate anticodons also read these codons to some extent. This background suppression leads to ‘statistical protein’ that contains some natural amino acid(s) at a site intended for NCAA. We characterize near-cognate suppression of amber, opal and a quadruplet codon in common Escherichia coli laboratory strains and find that the PylRS/tRNAPyl orthogonal pair cannot completely outcompete contamination by natural amino acids.Highlights► Nonsense suppression by natural amino acids hinders genetic code expansion. ► Higher levels of near-cognate suppression found in opal versus amber codons. ► E. coli BL21 and MG1655 display higher background suppression than TOP10. ► PylRS/tRNAUCAPyl cannot outcompete Trp incorporation at opal codons. ► PylRS/tRNAUCCUPyl is outcompeted by Arg-tRNAArg in quadruplet decoding.

Together with tRNACUAPyl, a rationally designed pyrrolysyl-tRNA synthetase mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivatives with large para substituents into superfolder green fluorescent protein at an amber mutation site in Escherichia coli. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivatives using engineered pyrrolysyl-tRNA synthetase-tRNACUAPyl pairs.

Using the amber suppression approach, four noncanonical amino acids (ncAAs) were used to replace existing amino acids at four positions in lasso peptide microcin J25 (MccJ25). The lasso peptide biosynthesis enzymes tolerated all four ncAAs and produced antibiotics with efficacy equivalent to wild-type in some cases. Given the rapid expansion of the genetically encoded ncAA pool, this study is the first to demonstrate an expedient method to significantly increase the chemical diversity of lasso peptides.

Correction for ‘Genetically encoded unstrained olefins for live cell labeling with tetrazine dyes’ by Yan-Jiun Lee et al., Chem. Commun., 2014, DOI: 10.1039/c4cc06435f.

Thirteen novel non-canonical amino acids were synthesized and tested for suppression of an amber codon using a mutant pyrrolysyl-tRNA synthetase–tRNAPylCUA pair. Suppression was observed with varied efficiencies. One non-canonical amino acid in particular contains an azide that can be applied for site-selective protein labeling.

The transient formation of nitrilimine in aqueous conditions is greatly influenced by pH and chloride. In basic conditions (pH 10) with no chloride, a diarylnitrilimine precursor readily ionizes to form diarylnitrilimine that reacts almost instantly with an acrylamide-containing protein and fluorescently labels it.

A number of non-canonical amino acids (NCAAs) with unstrained olefins are genetically encoded using mutant pyrrolysyl-tRNA synthetase–tRNAPylCUA pairs. These NCAAs readily undergo inverse electron-demand Diels–Alder cycloadditions with tetrazine dyes, leading to selective labeling of proteins bearing these NCAAs in live cells.

Using a mutant pyrrolysyl-tRNA synthetase-tRNAPylCUA pair, 3-formyl-phenylalanine is genetically incorporated into proteins at amber mutation sites in Escherichia coli. This non-canonical amino acid readily reacts with hydroxylamine dyes, leading to rapid and site-selective protein labelling.

Controlled orientation of a small laccase on a multi-walled carbon nanotube electrode was achieved via copper-free click chemistry mediated immobilization. Modification of the enzyme was limited to only the tethering site and involved the genetic incorporation of the unnatural amino acid 4-azido-L-phenylalanine (AzF). This approach enabled efficient direct electron transfer.

Fluorophenylalanines bearing 2–5 fluorine atoms at the phenyl ring have been genetically encoded by amber codon. Replacement of F59, a phenylalanine residue that is directly involved in interactions with trimethylated K9 of histone H3, in the Mpp8 chromodomain recombinantly with fluorophenylalanines significantly impairs the binding to a K9-trimethylated H3 peptide.