Wenshe R. Liu

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

We developed a general approach that allows unnatural amino acids with diverse physicochemical and biological properties to be genetically encoded in mammalian cells. A mutant Escherichia coli aminoacyl-tRNA synthetase (aaRS) is first evolved in yeast to selectively aminoacylate its tRNA with the unnatural amino acid of interest. This mutant aaRS together with an amber suppressor tRNA from Bacillus stearothermophilus is then used to site-specifically incorporate the unnatural amino acid into a protein in mammalian cells in response to an amber nonsense codon. We independently incorporated six unnatural amino acids into GFP expressed in CHO cells with efficiencies up to 1 μg protein per 2 × 107 cells; mass spectrometry confirmed a high translational fidelity for the unnatural amino acid. This methodology should facilitate the introduction of biological probes into proteins for cellular studies and may ultimately facilitate the synthesis of therapeutic proteins containing unnatural amino acids in mammalian cells.

Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation

Comparing the acetylomes with the phosphoproteomes of flies and humans suggests that phosphorylation sites may have evolved faster than did acetylation sites. Age of the Acetylome Acetylation and phosphorylation are regulatory posttranslational modifications that occur on proteins. With proteome-wide data in divergent species, insights regarding the evolution of these two regulatory processes can be revealed. Weinert et al. report the proteome-wide analysis of acetylated proteins in the fruit fly. Comparing the data on acetylated proteins in humans and flies with proteome sequences of nematodes and zebrafish indicated that acetylated sites were more conserved than were nonacetylated sites, and comparison of the human and fly acetylomes with their phosphoproteomes indicated that acetylation sites were more conserved than were phosphorylation sites. Acetylation intersected with another posttranslational modification, ubiquitylation: Acetylation occurred on one-third of human ubiquitin-conjugating E2 enzymes and influenced the activity of these enzymes, suggesting that acetylation provides another regulatory layer for this other type of posttranslational modification. Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines. With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines were significantly more conserved than were nonacetylated lysines. Bioinformatics analysis using Gene Ontology terms suggested that the proteins with conserved acetylation control cellular processes such as protein translation, protein folding, DNA packaging, and mitochondrial metabolism. We found that acetylation of ubiquitin-conjugating E2 enzymes was evolutionarily conserved, and mutation of a conserved acetylation site impaired the function of the human E2 enzyme UBE2D3. This systems-level analysis of comparative posttranslational modification showed that acetylation is an anciently conserved modification and suggests that phosphorylation sites may have evolved faster than acetylation sites.

Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA(Pyl). Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA(Pyl). These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.

Reversal of the hofmeister series: specific ion effects on peptides.

Ion-specific effects on salting-in and salting-out of proteins, protein denaturation, as well as enzymatic activity are typically rationalized in terms of the Hofmeister series. Here, we demonstrate by means of NMR spectroscopy and molecular dynamics simulations that the traditional explanation of the Hofmeister ordering of ions in terms of their bulk hydration properties is inadequate. Using triglycine as a model system, we show that the Hofmeister series for anions changes from a direct to a reversed series upon uncapping the N-terminus. Weakly hydrated anions, such as iodide and thiocyanate, interact with the peptide bond, while strongly hydrated anions like sulfate are repelled from it. In contrast, reversed order in interactions of anions is observed at the positively charged, uncapped N-terminus, and by analogy, this should also be the case at side chains of positively charged amino acids. These results demonstrate that the specific chemical and physical properties of peptides and proteins play a fundamental role in ion-specific effects. The present study thus provides a molecular rationalization of Hofmeister ordering for the anions. It also provides a route for tuning these interactions by titration or mutation of basic amino acid residues on the protein surface.

A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum.

Together with tRNA(CUA)(Pyl), 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-tRNA(CUA)(Pyl) pairs.

A Genetically Encoded Diazirine Photocrosslinker in Escherichia coli

Photoaffinity labels and crosslinkers have been used to map biomolecular interactions, as well as to identify the biological targets of small molecules. Benzophenones are among the most useful photocrosslinking agents and preferentially insert into C H bonds upon excitation with UV light. Aryl azides and [3-(trifluoromethyl)-3H-diazin-3yl]phenones generate reactive nitrenes and carbenes, respectively, that undergo relatively nonspecific insertion and addition reactions. 6] To facilitate the selective incorporation of photocrosslinking agents into proteins in living cells, we recently genetically encoded para-benzoyl-l-phenylalanine (pBpa) and para-azido-l-phenylalanine (pAzpa) in response to the amber nonsense codon in E. coli, yeast and mammalian cells. These photocrosslinkers were site-specifically incorporated into proteins by means of heterologous amber suppressor tRNA/aminoacyl–tRNA synthetase (aaRS) pairs that recognize the unnatural amino acid, but do not cross-react with endogenous host cell tRNAs, aaRSs or amino acids. To expand the photoaffinity label repertoire, we now report that 4’-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine (TfmdPhe; Figure 1A) can also be genetically encoded with excellent efficiency and fidelity in bacteria. TfmdPhe has useful photochemical properties and is stable under physiological conditions. Upon excitation by ~350 nm light, TfmdPhe undergoes fragmentation to N2 and a reactive carbene, which readily inserts into C H or O H bonds. In contrast, pAzpa requires relatively short wavelength UV excitation and can rearrange to less reactive secondary products prior to crosslinking. TfmdPhe is also somewhat smaller than pBpa, which can facilitate its incorporation into proteins at sites that are involved in biomolecular interactions. TfmdPhe has been incorporated into proteins previously by a chemically misacylated amber suppressor tRNA in a cell-free protein expression system. However, this method severely limits the protein yield, and is not amenable to studies directly in living cells. TfmdPhe was synthesized by using a previously reported method. To selectively incorporate TfmdPhe into proteins in E. coli, an orthogonal amber suppressor tRNA/aaRS was generated from a Methanococcus jannaschii amber suppressor tRNA (MjtRNACUA)/para-bromo-l-phenylalanyl-tRNA synthetase (BrPheRS) pair by using a previously reported, directed evolution strategy. BrPheRS was used as a template for mutagenesis because para-bromo-l-phenylalanine (BrPhe) is structurally similar to TfmdPhe, and BrPheRS has polypeptide backbone ACHTUNGTRENNUNGrearrangements that enlarge the substrate-binding pocket. Based on the structure of the BrPheRS–BrPhe complex, a library of aaRS active-site mutants was generated, in which residues L32, L65, H70, Q109, H160 and Y161 of BrPheRS were randomized by overlap extension polymerase chain reaction with synthetic oligonucleotide primers; the intended mutations were encoded by NNK (N=A+T+C+G; K=T+G). This library of aaRS mutants (on pBK plasmids under the control of the E. coli GlnRS promoter and terminator) was then passed through rounds of alternating positive and negative selections. In the positive selection, cell survival is dependent on the suppression of an amber codon that was introduced at a permissive site in the chloramphenicol acetyl transferase (CAT) gene in the presence of chloramphenicol and 1 mm TfmdPhe. The negative selection utilizes the toxic barnase gene with amber mutations at two permissive sites, and is carried out in the absence of TfmdPhe. Cells that contain MjTyrRS variants that acylate MjtRNACUA with TfmdPhe, but not any endogenous amino acids survive both positive and negative selections, whereas cells that contain MjTyrRS variants that acylate MjtRNACUA with endogenous amino acids express barnase and die in the negative selection. Five rounds of selection afforded five colonies that survived on chloramphenicol only in the presence of TfmdPhe. DNA sequencing of MjTyrRS mutants in these five colonies revealed one unique TfmdPheRS that had three new mutation sites : Y32I, H70F, and Q109M as well as four mutations (E107S, D158P, I159L, and L162E) that were inherited from BrPheRS. To determine the efficiency and fidelity of the incorporation of TfmdPhe into proteins, an amber codon was substituted for [a] Dr. E. M. Tippmann , Dr. W. Liu , Dr. D. Summerer, A. V. Mack, Prof. Dr. P. G. Schultz Department of Chemistry and the Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 N. Torrey Pines Rd, La Jolla, CA 92037 (USA) Fax: (+1)858-784-9440 E-mail : schultz@scripps.edu [b] Dr. E. M. Tippmann + Present address: Cardiff School of Chemistry, Cardiff University Cardiff, Wales, CF10 3XQ (UK) [c] Dr. W. Liu + Present address: Department of Chemistry, Texas A&M University College Station, TX 77842 (USA) [] These authors contributed equally to this work. Figure 1. A) Structure of 4’-[3-(trifluoromethyl)-3H-diazin-3-yl]-l-phenylalanine (TfmdPhe); B) Specificity and efficiency of TfmdPhe incorporation by TfmdPheRS–MjtRNA CUA pair. TfmdPhe was incorporated in response to an amber codon at position 7 in Z domain, analyzed by SDS-PAGE, and stained by gel code blue. Proteins were purified by Ni-affinity chromatography.

A Genetically Encoded Acrylamide Functionality

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.

A genetically encoded photocaged Nε-methyl-L-lysine

A photocaged N(epsilon)-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 N(epsilon)-methyl-L-lysine at physiological pH provide a convenient method for the biosynthesis of proteins with monomethylated lysines at specific sites.

A convenient method for genetic incorporation of multiple noncanonical amino acids into one protein in Escherichia coli

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 N(epsilon)-acetyl-l-lysines into one GFP(UV) protein in E. coli.

Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion

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.