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Incorporation of non-natural amino acids into proteins Takahiro Hohsaka and Masahiko Sisido* Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz’s group and Chamberlin’s group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.
codons, only the amber stop codon can be used for the suppression, multiple incorporation of non-natural amino acids into single proteins cannot be expected [4,5•].
Addresses Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan *e-mail:
[email protected]
Extension of codon–anticodon pairs
Current Opinion in Chemical Biology 2002, 6:809–815 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 18 October 2002 Abbreviations aaRS aminoacyl-tRNA synthetase aa-tRNA aminoacyl-tRNA DHFR dihydrofolate reductase
Introduction Amino acid substitution of proteins by genetic engineering had been restricted to the 20 naturally occurring amino acids. In 1989, the amber suppression method for introducing non-natural amino acids into specific positions of proteins was developed, opening the way to expanding protein functions [1,2]. In this method, amber suppressor tRNAs are aminoacylated with desired non-natural amino acids through chemical aminoacylation (i.e. an enzymatic ligation of an aminoacyl-dinucleotide with a tRNA lacking the terminal dinucleotide unit) [3]. The resulting aminoacyltRNAs (aa-tRNAs) are added to an in vitro translation system or Xenopus oocytes together with a mRNA or DNA of interest containing an amber stop codon (UAG) at a desired position. The amber codon is suppressed by the added aa-tRNA, resulting in the incorporation of the non-natural amino acid into the directed position of the target protein. This method, however, suffers from several disadvantages. First, stop codons are under tough competition with the release factors that terminate peptide elongation. Thus, the release factors interfere with the incorporation of nonnatural amino acids. Second, because among three stop
Preparation of aa-tRNAs is another issue to be improved, especially for a large-scale expression of non-natural proteins. Once an aa-tRNA is used for translation or is hydrolyzed at an aminoacyl bond, the deacylated tRNA will not be aminoacylated again in the cell-free translation systems or Xenopus oocytes. This disadvantage significantly decreases the yield of non-natural proteins. This review focuses on recent improvements of non-natural amino acid mutagenesis addressed to these problems. Applications of non-natural mutagenesis to create proteins with specialty functions are also introduced.
The genetic code consists of 61 codons for assigning 20 naturally occurring amino acids and 3 codons as stop signals. To fit non-natural amino acids into the existing genetic code system, it is essential to generate new codons that are specific to them. The use of an amber stop codon (Figure 1a) is one of the solutions without large alteration of the existing codon system. Alternatively, four-base codons have been developed (Figure 1b) [5•,6]. In an Escherichia coli in vitro translation system, various kinds of four-base codons — AGGU, CGGU, CGCU, CGAU, CCCU, CUCU, CUAU, GGGU, and their fourth-letter variants — were able to assign non-natural amino acids within the framework of the existing three-base codon system. Among them, GGGU gave the most efficient result, in which p-nitrophenylalanine (11, Figure 2) was introduced into streptavidin in 86% yield relative to a wildtype streptavidin. Because most of the four-base codons listed above are orthogonal to each other, double incorporation of two different non-natural amino acids into individual positions of streptavidin has been achieved by using efficient sets of the four-base codons such as GGGU and CGGG [5•]. Surprisingly, codons could be further extended to five-base codons such as CGGN1N2, where N1 and N2 indicate one of four nucleotides. Indeed, the five-base codons were decoded by aa-tRNAs containing the corresponding five-base anticodons [7•]. In addition, we have found that several kinds of four-base codons were also highly effective in a rabbit reticulocyte lysate (Hohsaka T, Sisido M, unpublished data). Magliery et al. undertook an in vivo selection of efficient four-base codons by a library method [8,9], and four four-base codons, AGGA, CUAG, UAGU and CCCU, were identified [8]. Another strategy for the extension of the genetic code has been proposed by using non-natural base pairs that are orthogonal to the naturally occurring four types. In 1991,
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Figure 1 (a) Amber codon
Non-natural amino acids discussed in the text.
(b) Four-base codon
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Bain et al. [10] showed that an isoC and isoG base pair is correctly formed when it is incorporated into codon and anticodon, respectively. Although the isoC–isoG pair works effectively in the translation process, the mRNA and tRNA containing the non-natural bases should be synthesized by chemical methods. Recently, novel non-natural base pairs that fit into the replication and transcription processes have been designed. Hirao et al. [11••,12] reported that a non-natural base, pyridine-2-one (y), was specifically
introduced into a mRNA corresponding to a 2-amino-6-(2thienyl)purine (s) in a DNA template. The non-natural codon yAG was successfully translated by a tRNA containing the corresponding non-natural anticodon CUs (Figure 1c) and aminoacylated with m-chlorotyrosine (22). Although the anticodon could not be introduced into the tRNA through transcription in this report, the result clearly demonstrates the feasibility of using a non-natural base pair to assign non-natural amino acids in in vitro and in vivo
Figure 2 legend Codons that can be assigned to non-natural amino acids. (a) Amber codon. An amber codon UAG is decoded by aa-tRNA containing a CUA anticodon. (b) Four-base codon. CGGG is shown as an example of four-base codons. The CGGG codon is decoded by aa-tRNA containing the corresponding
anticodon CCCG. (c) Non-natural codon. A recently developed non-natural codon-anticodon pair yAG-CUs and structures of y and s are shown. The yAG codon makes a pair with the CUs anticodon exclusively through a cellfree translation. The gray oval represents an amino acid.
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Figure 2 COOH
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transcription/translation systems. Hirao et al. [11••,12–14] designed non-natural base pairs incorporating an orthogonal hydrogen bond and steric hindrance. On the other hand, Romesberg and co-workers [15–17] have attempted to generate non-natural base pairs by introducing hydrophobic and stacking interactions into base pairs.
Aminoacylation of tRNAs with non-natural amino acids Because the chemical aminoacylation (Figure 3a) cannot be made in living cells, the non-natural mutagenesis had been carried out only in cell-free translation systems or in Xenopus oocytes through microinjection of the aa-tRNA. Exceptionally, amino acids that are analogous to natural ones are accepted as substrates by one of the aminoacyltRNA synthetases (aaRSs). For example, Kiick et al. [18•] found that a methionine analog, azidohomoalanine (33), was accepted by MetRS and incorporated into proteins in place of methionine in E. coli. The incorporated azido group was selectively modified by the Staudinger ligation with a phosphine-containing peptide. Recently, non-natural aminoacylation has been achieved by mutated aaRS (Figure 3b) by Schultz’s group. A pair of an amber suppressor tRNATyr and a TyrRS from Methanococcus jannaschii has been mutated to become orthogonal to any aaRS/tRNA pair from E. coli [19,20]. The latter TyrRS was further mutated not to accept tyrosine or any other amino acids, but to accept O-methyl-tyrosine (44) exclusively as the substrate [21••]. Five residues located in the active site of the TyrRS were randomly mutated. The library of the mutant TyrRSs was screened by two rounds of positive selection to accept O-methyl-tyrosine and subsequent negative selection to reject any natural amino acids. The final aaRS was found to accept O-methyl-tyrosine exclusively. Co-expression of the mutant TyrRS and amber suppressor tRNA in the presence of a dihydrofolate reductase (DHFR) gene containing an amber codon produced a mutant DHFR protein containing an O-methyltyrosine at the amber codon position in E. coli [21••]. An ion trap mass analysis demonstrated that O-methyl-tyrosine was incorporated at more than 95% fidelity. Similar approaches were carried out for 2-naphthylalanine (55) [22], p-azidophenylalanine (66) [23], and p-benzoylphenylalanine (77) [24••]. In addition, mutant aaRSs for p-aminophenylalanine (88) and p-isopropylphenylalanine (99) were also selected [25•]. Mutant aaRSs for non-natural amino acids with spin (110), fluorescent (111,12), and biotin labels (113), an aldehyde (114) or allyl (115) functionality, a metal binder (116), photocaged groups (117,18), a photoisomerizable group (119), and a sugar moiety (220) are being investigated [25•]. Hirao, Yokoyama and co-workers [26•] reported a mutated E. coli TyrRS that recognizes m-iodotyrosine (221) more efficiently than tyrosine. Three positions of the TyrRS were systematically mutated, and the aminoacylation activities
of each mutant with iodotyrosine and with tyrosine were compared by use of an amber suppressor E. coli tRNATyr. Then, two effective point mutations were combined together. One of these double mutants, V37C195, recognized iodotyrosine 10-fold more efficiently than it recognized tyrosine. Addition of the mutated enzyme and the amber suppressor tRNA to a wheat germ cell-free translation system expressing a c-Ha-Ras gene with an amber codon gave the mutant Ras protein containing an iodotyrosine at the directed position. LC-MS (liquid chromatography coupled with mass spectrometry) analysis demonstrated that iodotyrosine was incorporated in more than 95% fidelity. The wheat germ cell-free translation system has the advantage that eukaryotic proteins are liable to fold correctly. In addition, the E. coli TyrRS-tRNATyr pair was orthogonal to wheat germ tRNA-aaRS pairs without any mutagenesis. RajBhandary and co-workers [27,28] have developed a method for importing aminoacylated amber and ochre suppressor tRNAs into mammalian cells. They showed that both suppressor tRNAs aminoacylated with tyrosine were imported by using a transfecting reagent Effectene, and an active chloramphenicol acetyl transferase was expressed from co-transfected chloramphenicol acetyl transferase genes containing the amber or ochre codon at an internal position. Suga and co-workers [29–31] have reported aminoacylation of tRNAs with ribozymes. They screened a ribozyme from random combinatorial pools and aminoacylated tRNA with specific N-biotinylated amino acids including ε-biotinylated lysine (222, Figure 3c) [32••]. The aminoacylating ribozyme interacts with both the 3′ terminal and the anticodon loop of the target tRNA. The ribozyme strategy is not only useful for labeling of proteins with biotin, but is potentially applicable to customized ribozymes that aminoacylate desired non-natural amino acids to specific tRNAs.
Extension of protein functions with non-natural amino acids By introducing non-natural amino acids with specialty functions, protein functions will be widely expanded as described below. Dougherty’s group [33] have been investigating ion channels by using the amber suppression method in Xenopus oocytes. Caged amino acids, S-o-nitrobenzyl-cystein (223) and O-o-nitrobenzyl-tyrosine (117) have been introduced into nicotinic acetylcholine receptor and Kir2.1 channel [34,35]. The resulting receptors recovered their activity when the o-nitrobenzyl group was removed by UV irradiation. The receptor activity could be controlled also by introducing agonist-tethered amino acids (224) [36•]. Four kinds of O-(trimethylammoniumalkyl)tyrosine have been synthesized and incorporated at three different positions of the acetylcholine receptor. Because the trimethylammoniumalkyl group works as an agonist for the receptor, the resulting receptor–agonist conjugates showed constitutive activity in the absence of acetylcholine.
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Figure 3 Methods for aminoacylation of tRNAs with non-natural amino acids. (a) Chemical aminoacylation. A chemically synthesized aminoacyl-dinucleotide is enzymatically ligated by T4 RNA ligase with a tRNA lacking the terminal dinucleotide unit. (b) Aminoacylation by aaRSs generated by directed evolution to recognize both a non-natural amino acid and an anticodon of tRNA. (c) Aminoacylation by ribozymes that were screened by directed evolution. The ribozyme consists of acyltransfease and tRNA-binding domains. The 5′-OH of the ribozyme is aminoacylated by an aminoacylated pentaribonucleotide before the aminoacylation of 3′-OH of the tRNA. The gray oval represents an amino acid.
(a) Chemical aminoacylaion
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Current Opinion in Chemical Biology
Hecht and co-workers reported position-specific proteolysis by introducing allylglycine (225 ). Proteins containing allylglycine have been cleaved by treatment with iodine through a presumed iodolactone intermediate.
Introduction of allylglycine to a trypsinogen [37•] or a trypsin inhibitor (ecotin) [38] successfully controlled the trypsin activity. Non-natural amino acids with sugar moieties were also investigated by their group [39].
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Schultz and co-workers have been using their in vivo method to investigate protein–protein interactions in living cells. They incorporated p-benzoylphenylalanine (77) into a Phe52 position of dimeric glutathione S-transferase. Subsequent UV irradiation of the mutant resulted in a formation of a covalently linked homodimer [24••]. This photocrosslinking method will be useful for mapping protein–protein interactions in living cells.
Acknowledgements
Reversible photoregulation of proteins has been achieved by incorporating a photoisomerizable group such as azobenzene into specific positions. We have introduced p-phenylazophenylalanine (226) into various positions of a horseradish peroxidase [40]. The F179-mutant and F162mutant showed reversible photoregulation of the enzyme activity. Incorporation of phenylazophenylalanine into a camel anti-lysozyme antibody or into a DNA-binding protein also induced reversible photoresponse of ligand binding (Hohsaka T, Sisido M, unpublished data).
• of special interest •• of outstanding interest
Position-specific incorporation of fluorescent groups is an important step toward comprehensive proteome analysis. Because of the size limitation of non-natural amino acids that are accepted by the translation machinery, relatively small but long-wavelength emitting fluorescent amino acids have been searched for. Amino acids carrying coumarin (227) [41] and anthraniloyl (228) [42] group are in such class of amino acids. They can be incorporated into proteins in relatively high yields and their fluorescence intensity and wavelength are sensitive to the ligand binding. Larger fluorescent groups such as fluorescein had never been introduced into proteins. Very recently, however, a greenfluorescent BODIPY FL-labeled p-aminophenylalanine (229) was designed, synthesized and successfully incorporated into proteins through an E. coli cell-free translation system (Hohsaka T, Sisido M, unpublished data). This finding will open a way for practical application of the non-natural mutagenesis for comprehensive analysis of protein–protein and protein–nucleic acid interactions.
Conclusions Introduction of non-natural amino acids into proteins is a potential tool that is widely applicable to genomic, proteomic and cellular biological researches. Positionspecific incorporation of probes such as biotin and fluorescent groups will be useful for high-throughput analyses of protein–protein and protein–nucleic-acid interactions. The possibility is further expanded when multiple non-natural amino acids are incorporated by using the four-base codon strategy. For these purposes, cell-free protein synthesis is advantageous for its rapid and simple processing. In vivo synthesis of non-natural mutants using artificial aaRS is essential for large-scale expression and for analyses of protein–protein interactions in living cells. Large-scale synthesis is particularly required for utilization of the non-natural mutants as nano-sized chemical devices, such as biosensors, biophotoelectronic devices and protein microarrays.
Works on four-base codons was supported by the Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports and Culture, Japan (No. 11 102 003) and by Industrial Technology Research Grant Program in ’00 from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
1.
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2.
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3.
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4.
Cload ST, Liu DR, Froland AF, Schultz PG: Development of improved tRNAs for in vitro biosynthesis of proteins containing unnatural amino acids. Chem Biol 1996, 3:1033-1038.
5. •
Hohsaka T, Ashizuka Y, Taira H, Murakami H, Sisido M: Incorporation of nonnatural amino acids into proteins by using various fourbase codons in an Escherichia coli in vitro translation system. Biochemistry 2001, 40:11060-11064. Various kinds of four-base codons are developed. A highly efficient four-base codon pair for incorporation of two different non-natural amino acids into a single protein is also described. 6.
Sisido M, Hohsaka T: Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs. Appl Microbiol Biotechnol 2001, 57:274-281.
7. •
Hohsaka T, Ashizuka Y, Murakami H, Sisido M: Five-base codons for incorporation of nonnatural amino acids into proteins. Nucleic Acids Res 2001, 29:3646-3651. This is the first report that five-base codons are decoded by aa-tRNAs containing five-base anticodons in a translation system. Sixteen five-base codons are developed for incorporation of non-natural amino acids into proteins. 8.
Magliery TJ, Anderson JC, Schultz PG: Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of ‘shifty’ four-base codons with a library approach in Escherichia coli. J Mol Biol 2001, 307:755-769.
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Anderson JC, Magliery TJ, Schultz PG: Exploring the limits of codon and anticodon size. Chem Biol 2002, 9:237-244.
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Wang B, Brown KC, Lodder M, Craik CS, Hecht SM: Chemically mediated site-specific proteolysis. Alteration of protein–protein interaction. Biochemistry 2002, 41:2805-2813. This paper describes a useful strategy to cleave a polypeptide chain internally through an iodination of allylglycine incorporated at desired positions of proteins. 38. Baird T Jr, Wang B, Lodder M, Hecht SM, Craik CS: Generation of active trypsin by chemical cleavage. Tetrahedoron 2000, 56:9477-9485. 39. Fahmi NE, Golovine S, Wang B, Hecht SM: Studies toward the site specific incorporation of sugars into proteins: synthesis of glycosylated aminoacyl-tRNAs. Carbohydr Res 2001, 330:149-164. 40. Muranaka N, Hohsaka T, Sisido M: Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase. FEBS Lett 2002, 510:10-12. 41. Murakami H, Hohsaka T, Ashizuka Y, Hashimoto K, Sisido M: Site-directed incorporation of fluorescent nonnatural amino acids into streptavidin for highly sensitive detection of biotin. Biomacromolecules 2000, 1:118-125. 42. Taki M, Hohsaka T, Murakami H, Taira K, Sisido M: A non-natural amino acid for efficient incorporation into proteins as a sensitive fluorescent probe. FEBS Lett 2001, 507:35-38.
