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Adding New Chemistries to the Genetic Code ANRV413-BI79-15 ARI 3 May 2010 8:32 Adding New Chemistries to the Genetic Code Chang C. Liu and Peter G. Schultz The Scripps Research Institute, La Jolla, California 92037; email: [email protected], [email protected] by University of South Carolina - Columbia on 05/23/12. For personal use only. Annu. Rev. Biochem. 2010. 79:413–44 Annu. Rev. Biochem. 2010.79:413-444. Downloaded from www.annualreviews.org Key Words First published online as a Review in Advance on aminoacyl-tRNA synthetase, protein engineering, protein evolution, March 18, 2010 translation The Annual Review of Biochemistry is online at biochem.annualreviews.org Abstract This article’s doi: The development of new orthogonal aminoacyl-tRNA synthetase/ 10.1146/annurev.biochem.052308.105824 tRNA pairs has led to the addition of approximately 70 unnatural amino Copyright c 2010 by Annual Reviews. acids (UAAs) to the genetic codes of Escherichia coli, yeast, and mam- All rights reserved malian cells. These UAAs represent a wide range of structures and func- 0066-4154/10/0707-0413$20.00 tions not found in the canonical 20 amino acids and thus provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function. 413 ANRV413-BI79-15 ARI 3 May 2010 8:32 properties (3–8). A number of archaea and eu- Contents bacteria even encode the noncanonical amino acids selenocysteine or pyrrolysine for added INTRODUCTION .................. 414 function (9). Thus, the creation of organisms BACKGROUND..................... 414 with expanded genetic codes that include TRANSLATION WITH NEW amino acids beyond the common 20 building AMINO ACIDS ................... 416 blocks might allow the design of peptides and Encoding Unnatural Amino Acids in proteins with enhanced or novel activities. Escherichia coli ................... 417 Additional building blocks might also facilitate Encoding Unnatural Amino the incorporation of biophysical probes into Acids in Yeast ................... 423 proteins for the analysis or control of protein Encoding Unnatural Amino structure and function in vitro and in living Acids in Mammalian Cells ....... 424 cells. Finally, an expanded genetic code may Other Methods for Genetically provide an advantage in the evolution of new Encoding Unnatural molecular or organismal function. Here, we Amino Acids .................... 424 describe efforts to augment the genetic codes Recognition of Unnatural Amino of both prokaryotic and eukaryotic organisms Acids by Evolved with unnatural amino acids (UAAs) that have Aminoacyl-tRNA Synthetases . 425 novel properties, and we illustrate the utility Blank Codons...................... 426 of this methodology in exploring protein Optimized Systems structure and function and in generating for Protein Expression .......... 426 proteins with new and/or enhanced activities. GENETICALLY ENCODED UNNATURAL AMINO ACIDS AND THEIR APPLICATIONS . 427 BACKGROUND Probes of Protein Structure and Chemists have exquisite control over the struc- Function ....................... 428 tures of small molecules, but the control of Protein Therapeutics............... 430 macromolecular, especially protein, structure Protein Evolution with an and function still represents a major challenge. Expanded Genetic Code ........ 433 A number of approaches are being pursued CONCLUSION ..................... 434 to address this limitation. The first of these is solid-phase peptide synthesis, which allows chemists to rationally manipulate polypeptide structure using natural and unnatural amino INTRODUCTION acids (5, 10). Though this method is subject by University of South Carolina - Columbia on 05/23/12. For personal use only. Annu. Rev. Biochem. 2010.79:413-444. Downloaded from www.annualreviews.org Proteins carry out a remarkable range of to size limitations (peptides containing over functions—from photosynthesis to transcrip- 50–100 amino acids are difficult to synthesize tion and signal transduction—with only 20 in significant quantities), it can be used in amino acids. Nonetheless, proteins often conjunction with semisynthetic methods to PTM: require chemistries beyond those contained in produce larger full-length proteins containing posttranslational the canonical 20 amino acids for function, in- UAAs. For example, in native chemical ligation, modification cluding cofactors, such as pyridoxal, thiamine, a peptide containing a C-terminal α-thioester Unnatural amino flavins, and metal ions (1), and posttranslational is reacted with a second peptide containing an acid (UAA): an amino modifications (PTMs) such as methylation, N-terminal cysteine. The resulting thioester acid not specified by glycosylation, sulfation, and phosphorylation linkage undergoes an acyl rearrangement to the existing genetic code, which encodes (2). Moreover, the modification of proteins form a native peptide bond, joining the two only 20 amino acids by chemical methods can lead to new physic- smaller peptides (7). A C-terminal α-thioester ochemical, biological, or pharmacological leaving group can also be generated by an 414 Liu · Schultz ANRV413-BI79-15 ARI 3 May 2010 8:32 intein-mediated reaction arrested before the electronic, structural, and conformational final splicing step. This method, termed properties have been incorporated into ion expressed protein ligation (EPL), allows the channels directly in cells (35–37). This has aaRS: aminoacyl- linkage of a recombinantly expressed protein to allowed for detailed structure-function studies tRNA synthetase a synthetic UAA-containing peptide (8). Solid- by the systematic modification of individual phase peptide synthesis and ligation techniques protein residues (38), including the determi- have been used to modify protein backbones nation of the role of proline isomerization (11), make polymer-modified erythropoietin in channel gating (39). Nevertheless, these analogs (12, 13), introduce fluorescent probes methods are limited by the accessibility and into peptides and proteins, and produce stability of the aminoacyl-tRNA adducts, the modified signaling proteins, ion channels, and stoichiometric use of aminoacylated tRNAs histones (14–16). However, the general appli- that cannot be continuously delivered, and cation of these techniques is limited by the need the disruptive nature of microinjection and for protecting group chemistry, the restrictions transfection techniques. on the sites of ligation, the constraints on Another approach that is applicable to living protein folding, and the inherent extracellular cells involves the use of wild-type aminoacyl- nature of these synthetic techniques. tRNA synthetases (aaRSs) to incorporate UAAs Biosynthetic approaches have also been that are close structural analogs of canonical developed for the in vitro synthesis of pro- amino acids (40, 41). In this approach, a strain teins containing UAAs. In these techniques, auxotrophic for one of the common 20 amino truncated tRNAs are enzymatically ligated acids is used to substitute that amino acid to chemically aminoacylated nucleotides, with an UAA analog. Although the resulting effectively decoupling the identity of the tRNA wholesale replacement of a common amino from that of the attached amino acid (17–21). acid by an UAA cannot sustain exponential Cell-free translation systems then use these growth, nondividing cells are still viable and aminoacylated tRNAs for protein synthesis are able to overexpress proteins that contain in response to either “blank” (nonsense or the UAA. In these newly synthesized proteins, frameshift codons that can be used to specify the canonical amino acid is efficiently replaced an UAA) or coding codons. Though techni- by its UAA analog at all sites (42, 43). The cally challenging, this method has been very diversity in the range of UAA analogs that useful in the study of protein structure and can be incorporated using this approach has function. Examples include the incorporation been increased by aaRS overexpression (44), of amino acids with modified backbones (22, active-site engineering (45, 46), editing domain 23), fluorophores (24–26), functional groups mutations (47), and sorting by cell-surface corresponding to PTMs (27), photo- and display of the UAA whose incorporation was by University of South Carolina - Columbia on 05/23/12. For personal use only. Annu. Rev. Biochem. 2010.79:413-444. Downloaded from www.annualreviews.org chemically reactive side chains (28), and altered desired (48). The global incorporation of UAA pKas (29). This method has also been combined analogs by this method has a number of useful with mRNA display to generate selectable applications. For example, substitution of peptide libraries that contain UAAs (30, 31). methionine with selenomethionine introduces A variation of this approach involves the injec- a heavy atom into proteins for crystallographic tion or transfection of chemically or otherwise phasing experiments (49); replacement of me- aminoacylated tRNAs into living cells (32, thionine by noreleucine in cytochrome P450, 33). For example, by chemically acylating an leucine by 5,5,5-trifluoroleucine in chloram- amber suppressor tRNA, such as one derived phenicol acetyltransferase, and tryptophan by from Tetrahymena thermophilia tRNAGln (which 4-aminotryptophan in barstar and green fluo- affords high efficiency and fidelity of UAA rescent protein (GFP) yields proteins with new incorporation when microinjected into Xenopus activities and properties (50–53); and timed oocytes) (34), UAAs spanning a range of substitution of methionine or phenylalanine
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