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Proteins Carrying One or More Unnatural Amino Acids

and .

Proteins carrying unnatural amino acids with novel side chains add a new dimension to studies of protein structure and function. This chapter provides an overview of the various strategies that have been developed to date for the synthesis of such proteins.


Site-specific mutagenesis of DNA has been one of the most important advances in biology in the last twenty-five years. The site-specific replacement of an amino acid in a protein by any of the other nineteen amino acids has allowed investigations of the role of individual amino acids in folding, structure, stability, and function of a protein. In spite of the wealth of information that has been gathered, however, a limiting factor is that this technique can be used to replace an amino acid only with one of the other nineteen naturally occurring amino acids. The ability to introduce unnatural amino acids (amino acid analogues) with novel chemical, physical and biological properties either globally (at multiple sites) or at specific sites into proteins adds a new dimension to studies of protein structure and function. Analogues used include those that are photoactivatable or fluorescent, those that carry heavy atoms such as iodine, reactive side chains such as keto groups, spectroscopic probes, and those that mimic phospho-amino acids. Besides providing a method for the design of proteins with novel chemical and biological properties, proteins carrying such unnatural amino acids can be used for in vivo and in vitro studies on protein folding, structure, stability and function, protein-protein interactions and protein localization.

Most proteins contain twenty different amino acids, which are specified by sixty-one codons of the genetic code. The discovery of selenocysteine1,2and more recently pyrrolysine (4-methyl-pyrroline-5-carboxylate),3,4 has led to an extension of the genetic code to twenty-two amino acids. Selenocysteine found in prokaryotic and eukaryotic organisms is encoded by the stop codon UGA, whereas pyrrolysine discovered recently in some methanogenic archaea is encoded by the stop codon UAG. The work reviewed in here adds significantly to the number of amino acids that can be incorporated directly into proteins. Different strategies for global and site-specific incorporation of amino acid analogues into proteins both in vitro and in vivo have been developed (Fig. 1) allowing a wide range of potential applications of unnatural amino acid mutagenesis.

Figure 1. Proteins carrying one or more unnatural amino acids.

Figure 1

Proteins carrying one or more unnatural amino acids. Basic principles of global (A) and (B), and site-specific insertion of one (C) or two different (D) unnatural amino acids.

Global Incorporation of Amino Acid Analogues into Proteins in Vivo

Overexpression of Wild Type or Mutant Aminoacyl-tRNA Synthetases in E. coli Strains Auxotrophic for Certain Amino Acids tRNAs and aminoacyl-tRNA synthetases

During protein synthesis, tRNAs act as adapter molecules between the codons in the mRNA and the amino acids specified by the respective codons. The aminoacylation of tRNAs with their cognate amino acids is catalyzed by aminoacyl-tRNA synthetases in a two-step reaction.5,6 The aminoacyl-tRNA synthetase activates the amino acid to form an aminoacyl-adenylate intermediate and then transfers the amino acid to its cognate tRNA (Scheme 1):

Image ch80f1.jpg

Scheme 1. aa, amino acid; aaRS, aminoacyl-tRNA synthetase.

In general, there are twenty different aminoacyl-tRNA synthetases in a cell, each one specific for one of the twenty amino acids. The specificity of each enzyme for its cognate tRNA and amino acid is a key determinant of the specificity of the genetic code.7,10

Global replacement of amino acids by closely related analogues. The ability of the translational machinery to accommodate amino acid analogues, which are structurally similar to their natural counterparts, has long been known and exploited for random insertion of analogues into proteins in vivo (Fig. 1A). Many of the aminoacyl-tRNA synthetases activate closely related analogues of the natural amino acids and attach them to the cognate tRNA. Examples of these are: MetRS and selenomethionine,11 TrpRS and 5-hydroxytryptophan,12 PheRS and 3-fluorophenylalanine,13 LeuRS and azaleucine,14 ProRS and azetidine carboxylic acid.15 The incorporation of such analogues including a number of applications is reviewed in detail elsewhere.16-18 Particularly noteworthy is the replacement of all the methionines in a protein by selenomethionine for crystal structure analysis of proteins.11

More recently, Tirrell and coworkers have utilized the inherent permissiveness of Escherichia coli MetRS, LeuRS and IleRS to replace the corresponding amino acids in proteins with a wide range of analogues. The reduced activity of some of these aminoacyl-tRNA synthetases towards certain analogues used (e.g., trans-crotylglycine, hexafluoroleucine, trifluoroisoleucine) was overcome by overexpression of E. coli MetRS, LeuRS or IleRS, respectively.19-23 Proteins carrying some of the amino acid analogues have properties quite different from those of the wild type proteins. For example, a modified leucine zipper protein carrying trifluoroleucine in place of leucines was shown to have significantly increased thermal stability and higher resistance to chaotropic denaturants.22

A mutant of E. coli PheRS which can attach p-chlorophenylalanine to tRNAPhe has been isolated and characterized.24,25 The mutant PheRS (Ala294→Gly; Fig. 2), which has a larger amino acid binding pocket, still prefers phenylalanine over chlorophenylalanine and incorporation of chlorophenylalanine into protein is essentially random with the analogue partially replacing the normal amino acid. Using amino acid auxotroph strains of E. coli and overproduction of the appropriate aminoacyl-tRNA synthetase, however, Tirrell and coworkers have isolated some proteins in which an amino acid analogue replaces the normal amino acid almost completely. For example, overexpression of the aforementioned PheRS mutant using a phenylalanine auxotroph strain in minimal medium supplemented with analogues such as p-iodo-, p-cyano-, p-ethynyl- and p-azidophenylalanine and 2-, 3-, 4-pyridylalanine resulted in efficient insertion of these analogues into a test protein; the extent of analogue substitution for phenylalanine varying between 45 and 90% depending on the analogue.26 Based on the structural data available for Thermus thermophilus PheRS,27 the amino acid binding pocket of E. coli PheRS was enlarged further by additional mutagenesis (Ala294→Gly; Thr251→Gly; Fig. 2) leading to the successful incorporation into proteins in vivo of p-acetylphenylalanine carrying an aryl ketone functionality.28

Figure 2. Mutants of E.

Figure 2

Mutants of E. coli PheRS used to incorporate phenylalanine analogues into proteins. Schematic representation of interactions between PheRS and phenylalanine. PheRS-amino acid contacts are indicated by arrows. Black, E. coli PheRS; gray, T. thermophilus (more...)

In the experiments described above, the phenylalanine at each site in a protein is globally replaced by the respective phenylalanine analogues (Fig. 1A). In a clever approach, Kwon et al29 have exploited the degeneracy of the genetic code to replace only a subset of the phenylalanines with the amino acid analogue 2-naphthylalanine (Fig. 1B). Phenylalanine is encoded by two codons, UUU and UUC, which are read by a single phenylalanine tRNA with the anticodon GAA.30,31 The UUU codon is Proteins Carrying One or More Unnatural Amino Acids 3 recognized through a G⋅U wobble base interaction between the first nucleotide of the anticodon and the third nucleotide of the codon. A mutant yeast phenylalanine tRNA having the anticodon AAA, was introduced into E. coli cells to translate the UUU codon more efficiently through standard Watson-Crick interactions. In combination with a mutant form of yeast PheRS with relaxed amino acid specificity (Thr415→Gly; corresponding to the Thr251→Gly in E. coli PheRS), insertion of the analogue 2-naphthylalanine could be strongly biased towards the UUU codon (Fig. 1B). Re-assignment of degenerate codons represents an elegant way to restrict the incorporation of amino acid analogues to a specific codon, which is a complementary approach to strategies based on nonsense and frameshift suppression described below.

Use of Editing-Defective Mutants of Aminoacyl-tRNA Synthetases

The fidelity of protein biosynthesis is critically dependent on the accuracy of the aminoacylation reaction. Many aminoacyl-tRNA synthetases inherently lack the capacity to discriminate between closely related and naturally occurring amino acids and may misactivate those amino acids, which are similar and/or smaller in size and shape, at a frequency of 0.1 to 1%.32,33 Based on calculations of thermodynamic contributions, Pauling suggested that the discrimination of molecules that differ by only one methyl group, such as isoleucine and valine, will not exceed 100-150 fold.34 Therefore, many aminoacyl-tRNA synthetases possess a hydrolytic editing function or proofreading activity. Such aminoacyl-tRNA synthetases contain two catalytic sites, the first one for activation of the amino acid and a second one for editing of misactivated non-cognate amino acids35,36 or misaminoacylated tRNAs.37 Thus, the editing reaction can occur either prior to transfer of the activated amino acid (pretransfer editing) or following its transfer to the tRNA (posttransfer editing) (Scheme 2). Crystal structure analyses of several aminoacyl-tRNA synthetases have shown that the activation site and the editing site can be as much as 30 A apart.38,39

Image ch80f6.jpg

In particular, aminoacyl-tRNA synthetases that use aliphatic hydrophobic amino acids (e.g., valine, leucine, isoleucine) must distinguish among nearly isosteric substrates. A well characterized example for an aminoacyl-tRNA synthetase with editing activity is E. coli ValRS, which discriminates against closely related but non-cognate amino acids by a "double-sieve" mechanism, rejecting larger amino acids such as leucine and isoleucine at its synthetic active site and threonine at its editing site. Mutagenesis of residues involved in editing results in the accumulation of misaminoacylated tRNAs.35 Thus, disabling the editing function of a given aminoacyl-tRNA synthetase provides a method for misaminoacylation of tRNAs and thereby for introduction of unnatural amino acids into proteins in vivo.

Using such editing-defective mutants of E. coli ValRS, Marli, Schimmel and coworkers replaced more than 20% of valine in cellular proteins of E. coli randomly with amino butyric acid (Abu).40 Abu, a naturally occurring metabolite sterically similar to cysteine,41 is normally not incorporated into proteins. Although the similarities of the side chains between valine (isopropyl group) and Abu (ethyl group) lead to misactivation of Abu by ValRS, the misaminoacylated Abu-tRNAVal is usually eliminated by the subsequent editing function of ValRS. Random mutagenesis of the whole E. coli chromosome followed by stringent selection yielded mutant strains that misaminoacylated tRNAMVal with cysteine, and consequently with Abu.40 All mutations obtained were located in the editing domain of ValRS, which is highly conserved among different species (Fig. 3, A and B). Amino acid analysis of total cellular proteins of the E. coli strain bearing the Thr222→Pro mutation demonstrated clearly that almost 25% of the valines were replaced with Abu; it was shown that Abu was inserted specifically at valine codons.

Figure 3. Editing defective mutants of aminoacyl-tRNA synthetases.

Figure 3

Editing defective mutants of aminoacyl-tRNA synthetases. Schematic representation of the editing domain (CP1 domain; connecting peptide domain 1) located within some of the aminoacyl-tRNA synthetases (A). Alignment of the relevant sections within the (more...)

Similarly, attenuation of the editing activity of E. coli LeuRS allows incorporation of unnatural amino acids into proteins in vivo. Based on previous work by Martinis and co-workers,42,43 who identified a conserved threonine residue at position 252 of LeuRS as being important for its editing function (Fig. 3, A and C), a wide range of non-cognate amino acids such as norvaline and norleucine were inserted efficiently by overexpression of LeuRS carrying the Thr252→Tyr mutation.44

Site-Specific Insertion of Amino Acid Analogues into Proteins in Vitro

Approaches described so far involving the overexpression of aminoacyl-tRNA synthetase mutants with relaxed substrate specificity or editing defects are particularly suitable for the generation of proteins carrying global substitutions at multiple sites by an analogue for studies that normally require large amounts of protein such as NMR or X-ray crystallography. At the same time, the demand for proteins with modifications at specific sites has propelled the development of a variety of different approaches ranging from classical chemical synthesis and chemical modification to the more recent technologies employing expressed protein ligation and tRNA-mediated mutagenesis (nonsense suppression, 4- and 5-base frameshift suppression, use of unnatural base pairs).

Chemical Synthesis of Proteins and Site-Specific Chemical Modification

Methods based on chemical synthesis of peptides or chemical modification of specific unique residues in a protein that allow the site-specific incorporation of amino acid analogues into proteins in vitro include (i) total chemical synthesis, (ii) semi-synthesis involving the chemical or enzymatic ligation of a synthetic peptide fragment carrying the amino acid analogue to other peptides or proteins and (iii) chemical labeling through site-specific modification of a unique cysteine in a protein.45-47 While each of these approaches has proved useful in special cases, they all have certain limitations that have prevented their general use so far. For example, the approach based on total chemical synthesis requires that the chemically synthesized protein should fold into a functional molecule. It also puts a limit on the size of the protein that can be made using synthetic methodology. The site-specific cysteine modification requires either that the protein contains a single cysteine residue that is accessible for modification or a single cysteine residue that is uniquely reactive.

An important improvement in the semi-synthetic approach is a procedure developed by Muir and coworkers called "expressed protein ligation", in which a synthetic peptide carrying an amino acid analogue is ligated to the N- or C-terminus of a recombinant protein using a protein splicing reaction.48-50 This approach, reviewed in detail elsewhere,51 has been used for a variety of studies including the introduction, in vitro, of amino acid analogues with fluorescent groups at the N- and the C-terminal regions of proteins.

Nonsense Suppressor tRNA-Mediated Insertion of Amino Acid Analogues into Proteins Using Cell-Free Translation Systems

A general approach for the in vitro synthesis of proteins carrying amino acid analogues at specific sites has been developed by Schultz, Chamberlin and their coworkers based on earlier work of Hecht and colleagues.52-55 The approach is based on the suppression of an amber termination codon (UAG) at a predetermined site in the mRNA by an amber suppressor tRNA aminoacylated with the desired amino acid analogue (Figs. 1C and 4A). Protein synthesis is carried out in cell-free systems, ranging from standard total protein extract-based systems from prokaryotic (e.g., E. coli) and eukaryotic origin (e.g., wheat germ, rabbit reticulocyte) to fully reconstituted systems. The only requirement for this approach is that the suppressor tRNA, also described as "orthogonal" suppressor tRNA, is not recognized by aminoacyl-tRNA synthetases present in the respective cell-free systems. Otherwise, once the aminoacylated suppressor tRNA has inserted the amino acid analogue at the designated site in the target protein, it will be re-aminoacylated with a natural amino acid and insert the natural amino acid instead of the analogue, thus generating a heterogeneous pool of target protein molecules. The approach has been used successfully for analysis of protein structure and function in vitro.56-63 Recent improvements of protein yields generated in cell-free systems (reviewed in ref. 64) such as the continuous E. coli system developed by Spirin and co-workers65—now commercially available—and the continuous wheat germ system66 allow in vitro protein synthesis in scales of 10 — 100 milligrams and make this an attractive option to generate proteins carrying amino acid analogues at specific sites for in vitro studies.

Figure 4. Examples of codon-anticodon interactions utilized for site-specific incorporation of amino acid analogues.

Figure 4

Examples of codon-anticodon interactions utilized for site-specific incorporation of amino acid analogues. Use of amber suppressor tRNAs (A), frameshift suppressor tRNAs (B), and new codon-anticodon interactions (C) as illustrated by the unnatural y⋅s (more...)

Efforts to increase the yield of protein synthesis in bacterial cell-free systems which use amber suppressor tRNAs charged with amino acid analogues have also included attempts to increase the overall efficiency of suppression. Nonsense suppressor tRNAs have to compete against release factors which recognize stop codons and mediate translation termination.67 In E. coli, release factor RF1 is responsible for termination at UAG and UAA codons. Based on earlier studies which found that a thermosensitive mutation in E. coli RF1 leads to increased readthrough activity of UAG codons by suppressor tRNAs,68 Hecht and coworkers developed a modified S30 transcription/translation system with reduced RF1 levels.69 Partial inactivation of the temperature-sensitive RF1 mutant by mild heat-shock treatment improved the suppression efficiency of UAG by more than 10-fold and was particularly useful for the incorporation of charged and polar amino acid analogues that yield relatively low suppression efficiency.70,71 More recently, Ueda and co-workers described a bacterial cell-free system reconstituted from tagged recombinant protein factors purified to homogeneity.72 The system (termed "PURE") contains more than 30 components that were purified individually; including translation factors, aminoacyl-tRNA synthetases, methionyl-tRNA transformylase, tRNAs, ribosomes and T7 RNA polymerase. The omission of RF1 ensured high suppression efficiency of UAG codons and successful incorporation of an unnatural amino acid using an amber suppressor tRNA. Although the productivity of the PURE system is generally higher than that of a conventional E. coli system, it has yet to be established whether such a fully reconstituted system can be scaled up for large scale in vitro synthesis of proteins carrying amino acid analogues.

Suppressor tRNAs and Their Aminoacylation in Vitro

Isolation of suppressor tRNAs. The possibility to use improved cell-free systems for preparative scale synthesis of proteins carrying amino acid analogues highlights the need for the isolation and in vitro aminoacylation of orthogonal suppressor tRNAs. The suppressor tRNAs selected should be purified readily and in the large quantities necessary. Purification of suppressor tRNAs that are expressed in vivo in E. coli or eukaryotic cells can often be a difficult and time-consuming process involving a series of steps starting from extraction of total tRNA and subsequent isolation of the suppressor tRNA of interest by a combination of column chromatography and gel purification.73 An alternative is to use orthogonal suppressor tRNAs generated by in vitro transcription. A disadvantage, however, of using in vitro tRNA transcripts is the complete lack of base modifications, some of which are required for maximum activity and specificity of tRNAs. In particular, the modification of A37 next to the anticodon is known to be important for the activity of suppressor tRNAs by strengthening the interaction between codon and anticodon.74-76 It would therefore be desirable to introduce such modifications into in vitro tRNA transcripts prior to their use in cell-free systems.

In vitro aminoacylation of suppressor tRNA with amino acid analogues. The strategy developed by Hecht and coworkers and used by others for site-specific insertion of amino acid analogues into proteins in vitro consists of enzymatic ligation of the aminoacyl-dinucleotide pCpA~X (X=amino acid analogue) to the 3'-end of a suppressor tRNA transcript lacking the 3'-terminal pCpA sequence (Fig. 5A).52 An alternative strategy, that is also applicable to full length purified tRNAs isolated from cellular sources, could involve the removal of the 3'-terminal pA by E. coli RNase T.77 Use of this enzyme provides a ready source of suppressor tRNAs lacking the 3'-terminal pA, which could then be joined to pA~X (X=amino acid analogue) using RNA ligase.78 Alternatively, the natural tolerance of aminoacyl-tRNA synthetases towards amino acid analogues that are closely related to their cognate amino acids can be used to aminoacylate suppressor tRNAs with isotopically labeled amino acids through standard in vitro aminoacylation (Fig. 5B). For example, 2H- and 13C-labeled amino acids were incorporated site-specifically into proteins to demonstrate structural changes at the level of single amino acids in bacterioopsin during the photocycle.79,80

Figure 5. Strategies for attaching amino acid analogues to suppressor tRNAs in vitro.

Figure 5

Strategies for attaching amino acid analogues to suppressor tRNAs in vitro. Chemical aminoacylation of a dpCpA dinucleotide followed by RNA ligase catalyzed joining of the aminoacyl-dpCpA to amber suppressor tRNA lacking the 3'-terminal pCpA (A), aminoacyl-tRNA (more...)

Recent work by Suga and colleagues demonstrates the potential of ribozyme-mediated aminoacylation of suppressor tRNAs in vitro with selected phenylalanine and tyrosine analogues (Fig. 5C). In vitro selection was used to generate bifunctional ribozymes that specifically recognize an activated amino acid and aminoacylate a tRNA. Such ribozymes, which execute the key functions of a typical aminoacyl-tRNA synthetase, consist of two different domains: the first domain interacts with the activated amino acid leading to self-aminoacylation of the 5'-hydroxyl group of the ribozyme; the second domain catalyzes the transfer of the aminoacyl group to the 3' end of the tRNA.81,82 A resin-immobilized form of ribozymes that catalyze the aminoacylation of suppressor tRNA with various phenylalanine and tyrosine analogues was developed providing a potentially powerful alternative for the generation of some aminoacyl-tRNAs.83

New Codon-Anticodon Pairs

Use of 4- and 5-base codon-anticodon interactions. Site-specific incorporation of two different unnatural amino acids into a protein requires further expansion of the genetic code and an extension of the standard codon-anticodon pair. Based on earlier work of Riddle & Roth84 and Yourno85 who initially demonstrated that a codon consisting of four bases could be translated by a mutant tRNA containing an extra nucleotide in the anticodon (frameshift suppressor tRNA), Sisido and coworkers have used four- and five-base codons for the site-specific insertion of one or two different amino acid analogues into proteins in vitro (Fig. 4B).86-89 Taking advantage of the fact that some codons are rarely used in bacteria and in eukaryotes, frameshift suppressor tRNAs decoding such four- and five-base codons were designed to work efficiently without serious competition from endogenous tRNAs present in prokaryotic and eukaryotic cell-free translation systems. The successful incorporation of various aromatic amino acid analogues, some carrying relatively large and bulky side groups, demonstrated the adaptability of the translational apparatus towards amino acid analogues.87 The combination of two different frameshift suppressor tRNAs that are highly specific for their respective complementary four-base codons allowed the site-specific insertion in vitro of two different amino acid analogues into two different sites of a single protein.90 Similarly, Hecht and coworkers have used an amber suppressor tRNA along with a frameshift suppressor tRNA to synthesize in vitro a protein containing two different amino acid analogues.91

Use of novel unnatural base pairs. Yet another strategy developed by Benner, Chamberlin, Yokoyama and coworkers involves expansion of the genetic code through the use of a 65th codon-anticodon pair, e.g., isoC⋅isoG, based on unnatural nucleoside bases with non-standard base pairing.92-94 Site-specific insertion of 3-iodotyrosine into a peptide was demonstrated using an in vitro translation system supplemented with a chemically synthesized mRNA containing the modified (isoC)AG codon at the site of interest and a suppressor tRNA containing the complementary CU(isoG) anticodon. To overcome the shortcomings of this approach, mainly caused by the requirement for chemical synthesis of both mRNA and tRNA, Yokoyama and co-workers have developed new unnatural bases that can be incorporated into RNA through in vitro transcription by T7 RNA polymerase.93,94 For example, the unnatural base pyridin-2-one (y) was inserted into mRNA in response to bases 2-amino-6-methylaminopurine (x) or 2-amino-6-(2-thienyl)purine (s) in the template DNA (Fig. 4C). These base pairs, y⋅x and y⋅s, which were designed on the basis of hydrogen-bonding pattern and shape complementarity, show high specificity in transcription and were used in a coupled transcription-translation system for site-specific insertion of amino acid analogues. In addition, novel unnatural base pairs that are also accepted by the replication machinery have been reported to increase the efficiency further by amplification of the DNA template.95-97 Similarly, the groups of Schultz and Romesberg have used the concept of hydrophobic bases as building blocks for novel base pairs.98-101

Site-Specific Incorporation of Amino Acid Analogues into Proteins in Vivo

The availability of methods to incorporate amino acid analogues site-specifically into proteins in vivo in bacteria and eukaryotes greatly expands the scope of unnatural amino acid mutagenesis. First, there is the potential to synthesize large amounts of the protein, making this a particularly useful technique for preparing material for sample-intensive methods. Second, potential problems associated with post-translational modifications and folding may be overcome for some proteins if a eukaryotic system is used. And third, the availability of an in vivo system opens the door to in vivo structure-function studies including protein folding and stability, protein dynamics, protein localization, protein-protein interactions, and analysis of signal transduction pathways.

Use of "Orthogonal" Aminoacyl-tRNA Synthetase/Suppressor tRNA Pairs

As for the in vitro system, the basic strategy relies upon translation of a mRNA carrying an amber mutation at a predetermined site using an amber suppressor tRNA that is aminoacylated with the amino acid analogue (Fig. 1C). The key requirements for the in vivo system are: (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases and (ii) an aminoacyl-tRNA synthetase that specifically recognizes the suppressor tRNA but no other tRNA in the cell. Because most cells contain twenty aminoacyl-tRNA synthetases in the cytoplasm, such a new aminoacyl-tRNA synthetase/suppressor tRNA pair represents a 21st (orthogonal) synthetase/tRNA pair.102-104 The next requirement is generation of mutants of the aminoacyl-tRNA synthetase, which activate the amino acid analogue instead of the natural amino acid and attach it to the orthogonal suppressor tRNA (Fig. 6A).

Figure 6. Strategies for site-specific incorporation of amino acid analogues in vivo.

Figure 6

Strategies for site-specific incorporation of amino acid analogues in vivo. Use of 21st (orthogonal) aminoacyl-tRNA synthetase/suppressor tRNA pairs (A); import of aminoacylated suppressor tRNAs into cells by microinjection, electroporation and transfection (more...)

A growing number of orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pairs have been identified so far, based mostly on the species-specificity of certain aminoacyl-tRNA synthetases for their respective tRNAs.105-108For example, Saccharomyces cerevisiae TyrRS mutants can be used to aminoacylate an amber suppressor tRNA derived from the E. coli initiator tRNA2fMet for possible insertion of unnatural amino acids into proteins in E. coli.104,105 Similarly, E. coli GlnRS can be used in eukaryotic cells to aminoacylate amber suppressor tRNAs derived from either the human initiator tRNAiMet or E. coli tRNAGln.102,104,109 Based on a TyrRS/tRNATyr pair from the archaeon Methanococcus jannaschii, Schultz and coworkers have identified a different orthogonal aminoacyl-tRNA synthetase/suppressor tRNA pair for use in E. coli.110-112 A M. jannaschii amber suppressor tRNATyr library was generated and passed through extensive negative and positive selection schemes, to discard those tRNA mutants that were recognized by E. coli aminoacyl-tRNA synthetases and to retain those mutants that are still efficiently aminoacylated by the M. jannaschii TyrRS. The resulting suppressor tRNATyr was then used to screen a library of M. jannaschii TyrRS mutants carrying random changes around binding pocket for the tyrosine side chain. Following several rounds of positive and negative selections, M. jannaschii TyrRS mutants which use analogues such as O-methyltyrosine, p-azidophenylalanine, 2-naphthylalanine and p-acetylphenylalanine instead of tyrosine and allow in vivo incorporation of those amino acid analogues into proteins in E. coli were obtained. Using a similar strategy, mutants of E. coli TyrRS that allow site-specific incorporation of five different amino acid analogues into proteins in yeast were identified.113

Yokoyama and coworkers have used an orthogonal pair based on E. coli TyrRS and Bacillus stearothermophilus suppressor tRNATyr for site-specific insertion of 3-iodotyrosine into proteins in mammalian cells.114,115 An E. coli TyrRS mutant with increased affinity for the amino acid analogue was engineered based on the known crystal structure of B. stearothermophilus TyrRS.116 To accommodate the bulky iodine substitution of 3-iodotyrosine, residues Tyr37, Gln179 and Gln195 in the tyrosine binding site were mutated individually or in combination and screened for activation of 3-iodotyrosine by in vitro biochemical assay. A combination of mutations Tyr37→Val and Gln195→Cys yielded a TyrRS variant which activates 3-iodotyrosine 10-fold more efficiently than tyrosine.114 When supplied in the growth medium of mammalian cells, 3-iodotyrosine was incorporated at a predetermined site into a reporter protein to >95%. In addition, the efficiency of suppression was significantly improved when the suppressor tRNATyr gene was expressed from a gene cluster, in which the tRNA gene was tandemly repeated several times.115

Approaches not Involving Orthogonal Aminoacyl-tRNA Synthetases: Microinjection, Microelectroporation, or Transfection of Aminoacylated Suppressor tRNAs into Oocytes and Mammalian Cells

The above approach utilizing aminoacyl-tRNA synthetase/suppressor tRNA pairs that are orthogonal to the host cells requires the isolation, one at a time, of different synthetase mutants for each amino acid analogue to be used. An alternative system, that does not involve a mutant aminoacyl-tRNA synthetase and that has the potential of being generally applicable would be the import into cells (by injection, transfection or electroporation) of suppressor tRNAs aminoacylated in vitro with the amino acid analogue of choice (Fig. 6B).73,117-120 The only requirement is that the suppressor tRNA must not be aminoacylated by any of the aminoacyl-tRNA synthetases in the cell. This approach is quite flexible in that the same suppressor tRNA can be chemically aminoacylated with virtually any amino acid analogue in vitro, while the synthesis of the protein of interest is performed in vivo.

An important step along these lines has been taken by Dougherty, Lester and their coworkers, who have injected chemically aminoacylated suppressor tRNAs into individual Xenopus oocytes for the site-specific insertion of amino acid analogues into membrane receptor and ion channel proteins.117,121 The Xenopus oocyte represents a well-established system for heterologous gene expression and subsequent characterization of proteins and was used to gain insight into the structure-function of ligand-gated ion channels, such as the nicotinic acetylcholine receptor. Amino acid analogues with different hydrophobic character were used to study the effect of polarity/hydrophobicity on the gating mechanism of such a cation-selective channel.122 Photochemical modifications using analogues with fluorescent or photoactivatable moieties established the importance of aromatic residues (tryptophan and tyrosine) for binding of the agonist acetylcholine;123,124 furthermore, the cation-π interaction between agonist and receptor was probed in detail with a series of fluorinated tryptophan analogues based on the fact that fluorine has a significant and additive effect on cation-π interaction.125-128

The "import" of aminoacylated suppressor tRNAs by means of transfection offers a new and versatile approach to site-specific insertion of one or two different unnatural amino acids into proteins in mammalian cells.73,120 Advances in modern transfection technologies allow the efficient delivery of purified suppressor tRNAs to a variety of different cell types and—in a single experiment—to a large number of cells limited only by the overall transfection efficiency. Similarly, microinjection and electroporation have also been used to deliver purified suppressor tRNAs into mammalian cells providing a more relevant environment for many cell-type specific analyses.118,119

We previously described the import of purified suppressor tRNAs into mammalian cells and the identification of an amber suppressor tRNA (supF) derived from E. coli tyrosine tRNA suitable for insertion of amino acid analogues into proteins in mammalian cells.73 In further extension of this work, an orthogonal ochre suppressor tRNA (supC.A32), also derived from E. coli tyrosine tRNA, was identified. The import of a mixture of supF amber and supC.A32 ochre suppressor tRNAs aminoacylated in vitro prior to transfection, led to concomitant suppression of an amber and an ochre codon in a single mRNA (Fig. 1D)120, whereas import of the same tRNAs without prior aminoacylation did not. These results represent the first report of successful suppression in vivo of two different nonsense codons in a single mRNA in a eukaryotic cell. They also provide a general approach to introduction of two different amino acid analogues into a protein. The possibility of incorporating two different amino acid analogues into a mammalian protein greatly increases the scope of unnatural amino acid mutagenesis. For example, introduction of two different fluorescent amino acids would allow the use of fluorescence resonance energy transfer to study protein conformation and dynamics in mammalian cells. Similarly, site-specific insertion of phospho-amino acids such as phosphothreonine and phosphotyrosine could be employed to activate a specific component within a signal transduction pathway, e.g., one of the many mitogen activated protein kinases, in the absence of an extracellular or upstream signal.

Summary and Perspectives

The incorporation of unnatural amino acids into proteins to generate proteins with novel biochemical and biophysical properties has emerged as a powerful tool for studying protein structure and function, as well as designing new proteins. Strategies based on the chemical synthesis of proteins carrying unnatural amino acids are complemented by template-directed incorporation of unnatural amino acids in cell-free systems of prokaryotic and eukaryotic origin. Amber and frameshift suppressor tRNAs aminoacylated in vitro with unnatural amino acids have been used for cell-free synthesis of proteins carrying one or more unnatural amino acids at specific sites. While synthesized only in limited amounts so far, studies of such proteins have provided significant new information on protein folding, structure and function. The recent development and commercialization of highly efficient cell-free systems amenable to scale up make possible the production of much larger amounts and, thereby, wider use of such proteins.

Overproduction of wild type or mutant aminoacyl-tRNA synthetases has led to the production in vivo of proteins in which a particular amino acid is globally replaced by one of the analogues throughout the protein. A key strategy for site-specific incorporation of an unnatural amino acid into a protein in vivo involves the readthrough of an amber stop codon by an amber suppressor tRNA that is aminoacylated in vivo with the unnatural amino acid. This approach has been used successfully for the site-specific incorporation of unnatural amino acids into proteins in E. coli, yeast and mammalian cells. A more general strategy involves the import of suppressor tRNAs aminoacylated in vitro with the unnatural amino acid into mammalian cells by injection, transfection, or electroporation. Recent work on this, involving the import of amber and ochre suppressor tRNAs, should allow the site-specific insertion of two different unnatural amino acids into a protein in mammalian cells and greatly expands the scope and applications of unnatural amino acid mutagenesis.


Work in our laboratory is supported by grants DAAD 19-99-1-0300 from the U.S. Army Research Office and GM17151 from the National Institutes of Health.


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