Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Designer proteins: applications of genetic code expansion in cell biology

Nature Reviews Molecular Cell Biology volume 13, pages 168–182 (2012)Cite this article

Subjects

Key Points

  • A large number of unnatural amino acids can now be incorporated into proteins using 'orthogonal' aminoacyl-tRNA synthetase–tRNA pairs. The pyrrolysyl-tRNA synthetase (PylRS)–tRNACUA pair is currently the most versatile, and it has been used in bacteria, yeast, mammalian cells and Caenorhabditis elegans.

  • Photocrosslinking amino acids allows protein interactions to be defined in vitro and in bacteria, yeast and mammalian cells.

  • Post-translational modifications, including lysine acetylation, monomethylation and dimethylation, and ubiquitylation, can be quantitatively directed into proteins by genetic code expansion.

  • Photocaged amino acids allow rapid activation of protein function inside living cells. This approach can be used to dissect signalling pathways.

  • Biophysical probes can be incorporated into proteins. Infrared probes have been used to examine conformational changes in G protein-coupled receptors. Other probes will probably have similar uses.

  • Bio-orthogonal chemistry provides a promising approach for labelling proteins for a range of applications, including imaging, but more rapid methods are needed for cellular imaging.

  • More sophisticated methods for incorporating multiple distinct amino acids, including orthogonal ribosome evolution and the generation of new synthetase–tRNA pairs are likely to expand the range of applications that are possible in the future.

Abstract

Designer amino acids, beyond the canonical 20 that are normally used by cells, can now be site-specifically encoded into proteins in cells and organisms. This is achieved using 'orthogonal' aminoacyl-tRNA synthetase–tRNA pairs that direct amino acid incorporation in response to an amber stop codon (UAG) placed in a gene of interest. Using this approach, it is now possible to study biology in vitro and in vivo with an increased level of molecular precision. This has allowed new biological insights into protein conformational changes, protein interactions, elementary processes in signal transduction and the role of post-translational modifications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expanding the genetic code.
Figure 2: In vivo photocrosslinking of membrane proteins and study of protein interactions.
Figure 3: Applications of genetically encoded post-translational modifications.
Figure 4: Genetically encoded photocaged amino acids allow insights into cell biology in real time.
Figure 5: Use of genetically encoded infrared probes to dissect local GPCR conformational changes during activation.

Similar content being viewed by others

References

  1. Johnson, J. A., Lu, Y. Y., Van Deventer, J. A. & Tirrell, D. A. Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications. Curr. Opin. Chem. Biol. 14, 774–780 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Xie, J. & Schultz, P. G. A chemical toolkit for proteins — an expanded genetic code. Nature Rev. Mol. Cell Biol. 7, 775–782 (2006).

    Article  CAS  Google Scholar 

  3. Chin, J. W. et al. An expanded eukaryotic genetic code. Science 301, 964–967 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Hancock, S. M., Uprety, R., Deiters, A. & Chin, J. W. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J. Am. Chem. Soc. 132, 14819–14824 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mukai, T. et al. Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818–822 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Genetically encoding Nɛ-acetyllysine in recombinant proteins. Nature Chem. Biol. 4, 232–234 (2008). Demonstrates, for the first time, that Lys acetylation can be site-specifically encoded in proteins.

    Article  CAS  Google Scholar 

  7. Gautier, A. et al. Genetically encoded photocontrol of protein localization in mammalian cells. J. Am. Chem. Soc. 132, 4086–4088 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Xie, J. M. & Schultz, P. G. An expanding genetic code. Methods 36, 227–238 (2005).

    Article  CAS  PubMed  Google Scholar 

  9. Chin, J. W., Martin, A. B., King, D. S., Wang, L. & Schultz, P. G. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl Acad. Sci. USA 99, 11020–11024 (2002). Describes the first genetically encoded photocrosslinking between the monomers of a glutathione S -transferasedimer in E. coli.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wu, N., Deiters, A., Cropp, T. A., King, D. & Schultz, P. G. A genetically encoded photocaged amino acid. J. Am. Chem. Soc. 126, 14306–14307 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Greiss, S. & Chin, J. W. Expanding the genetic code of an animal. J. Am. Chem. Soc. 133, 14196–14199 (2011). Shows that it is possible to use genetic code expansion to specifically modify a protein in an animal.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chin, J. W. et al. Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124, 9026–9027 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Ai, H.-W., Shen, W., Sagi, A., Chen, P. R. & Schultz, P. G. Probing protein–protein interactions with a genetically encoded photo-crosslinking amino acid. Chembiochem 12, 1854–1857 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Chou, C., Uprety, R., Davis, L., Chin, J. W. & Deiters, A. Genetically encoding an aliphatic diazirine for protein photocrosslinking. Chem. Sci. 2, 480–483 (2011).

    Article  CAS  Google Scholar 

  15. Tippmann, E. M., Liu, W., Summerer, D., Mack, A. V. & Schultz, P. G. A genetically encoded diazirine photocrosslinker in Escherichia coli. Chembiochem 8, 2210–2214 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Zhang, M. et al. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. Naure Chem. Biol. 7, 671–677 (2011).

    Article  CAS  Google Scholar 

  17. Braig, D., Bar, C., Thumfart, J. O. & Koch, H. G. Two cooperating helices constitute the lipid-binding domain of the bacterial SRP receptor. J. Mol. Biol. 390, 401–413 (2009).

    Article  CAS  PubMed  Google Scholar 

  18. Hino, N. et al. Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid. Nature Methods 2, 201–206 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Ieva, R. & Bernstein, H. D. Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane. Proc. Natl Acad. Sci. USA 106, 19120–19125 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mori, H. & Ito, K. Different modes of SecY–SecA interactions revealed by site-directed in vivo photo-cross-linking. Proc. Natl Acad. Sci. USA 103, 16159–16164 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Okuda, S. & Tokuda, H. Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB. Proc. Natl Acad. Sci. USA 106, 5877–5882 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kaiser, C. M. et al. Real-time observation of trigger factor function on translating ribosomes. Nature 444, 455–460 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Lakshmipathy, S. K. et al. Identification of nascent chain interaction sites on trigger factor. J. Biol. Chem. 282, 12186–12193 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Haslberger, T. et al. M domains couple the ClpB threading motor with the DnaK chaperone activity. Mol. Cell 25, 247–260 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Schlieker, C. et al. Substrate recognition by the AAA+ chaperone ClpB. Nature Struct. Mol. Biol. 11, 607–615 (2004).

    Article  CAS  Google Scholar 

  26. Liu, C. et al. Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature 463, 197–202 (2010).

    Article  CAS  PubMed  Google Scholar 

  27. Boos, D., Kuffer, C., Lenobel, R., Korner, R. & Stemmann, O. Phosphorylation-dependent binding of cyclin B1 to a Cdc6-like domain of human separase. J. Biol. Chem. 283, 816–823 (2008).

    Article  CAS  PubMed  Google Scholar 

  28. Tagami, S. et al. Crystal structure of bacterial RNA polymerase bound with a transcription inhibitor protein. Nature 468, 978–982 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Mohibullah, N. & Hahn, S. Site-specific cross-linking of TBP in vivo and in vitro reveals a direct functional interaction with the SAGA subunit Spt3. Genes Dev. 22, 2994–3006 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen, H. T., Warfield, L. & Hahn, S. The positions of TFIIF and TFIIE in the RNA polymerase II transcription preinitiation complex. Nature Struct. Mol. Biol. 14, 696–703 (2007).

    Article  CAS  Google Scholar 

  31. Yamano, K., Tanaka-Yamano, S. & Endo, T. Tom7 regulates Mdm10-mediated assembly of the mitochondrial import channel protein Tom40. J. Biol. Chem. 285, 41222–41231 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Carvalho, P., Stanley, A. M. & Rapoport, T. A. Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p. Cell 143, 579–591 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hino, N. et al. Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells. J. Mol. Biol. 406, 343–353 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Zimmer, J., Nam, Y. & Rapoport, T. A. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature 455, 936–943 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Mann, M. & Jensen, O. N. Proteomic analysis of post-translational modifications. Nature Biotech. 21, 255–261 (2003).

    Article  CAS  Google Scholar 

  36. Neumann, H., Hazen, J. L., Weinstein, J., Mehl, R. A. & Chin, J. W. Genetically encoding protein oxidative damage. J. Am. Chem. Soc. 130, 4028–4033 (2008).

    Article  CAS  PubMed  Google Scholar 

  37. Liu, C. C. & Schultz, P. G. Recombinant expression of selectively sulfated proteins in Escherichia coli. Nature Biotech. 24, 1436–1440 (2006).

    Article  CAS  Google Scholar 

  38. Serwa, R. et al. Chemoselective staudinger-phosphite reaction of azides for the phosphorylation of proteins. Angew. Chem. Int. Ed. Engl. 48, 8234–8239 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Xie, J. M., Supekova, L. & Schultz, P. G. A genetically encoded metabolically stable analogue of phosphotyrosine in Escherichia coli. ACS Chem. Biol. 2, 474–478 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Park, H.-S. et al. Expanding the genetic code of Escherichia coli with phosphoserine. Science 333, 1151–1154 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ai, H. W., Lee, J. W. & Schultz, P. G. A method to site-specifically introduce methyllysine into proteins in E. coli. Chem. Commun. (Camb.) 46, 5506–5508 (2010).

    Article  CAS  Google Scholar 

  42. Groff, D., Chen, P. R., Peters, F. B. & Schultz, P. G. A genetically encoded epsilon-N-methyl lysine in mammalian cells. Chembiochem 11, 1066–1068 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang, Y. S. et al. A genetically encoded photocaged Nɛ-methyl-L-lysine. Mol. Biosyst. 6, 1557–1560 (2010).

    Article  CAS  PubMed  Google Scholar 

  44. Nguyen, D. P., Garcia Alai, M. M., Kapadnis, P. B., Neumann, H. & Chin, J. W. Genetically encoding Nɛ-methyl-L-lysine in recombinant histones. J. Am. Chem. Soc. 131, 14194–14195 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Nguyen, D. P., Garcia Alai, M. M., Virdee, S. & Chin, J. W. Genetically directing ɛ-N, N-dimethyl-L-lysine in recombinant histones. Chem. Biol. 17, 1072–1076 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Virdee, S. et al. Traceless and site-specific ubiquitination of recombinant proteins. J. Am. Chem. Soc. 133, 10708–10711 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Virdee, S., Ye, Y., Nguyen, D. P., Komander, D. & Chin, J. W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nature Chem. Biol. 6, 750–757 (2010). References 46 and 47 demonstrate the site-specific ubiquitylation of recombinant proteins. Reference 47 also demonstrates the potential of this approach for elucidating the specificity of deubiquitinases and solving the structures of ubiquitylated proteins.

    Article  CAS  Google Scholar 

  48. Li, X., Fekner, T., Ottesen, J. J. & Chan, M. K. A pyrrolysine analogue for site-specific protein ubiquitination. Angew. Chem. Int. Ed. Engl. 48, 9184–9187 (2009).

    Article  CAS  PubMed  Google Scholar 

  49. Liu, C. C. et al. Protein evolution with an expanded genetic code. Proc. Natl Acad. Sci. USA 105, 17688–17693 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000).

    Article  CAS  PubMed  Google Scholar 

  51. Kim, J. et al. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7, 397–403 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Fischle, W. et al. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev. 17, 1870–1881 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kouzarides, T. Chromatin modifications and their function. Cell 128, 693–705 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001).

    Article  CAS  PubMed  Google Scholar 

  55. Zhao, S. M. et al. Regulation of cellular metabolism by protein lysine acetylation. Science 327, 1000–1004 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834–840 (2009).

    Article  CAS  PubMed  Google Scholar 

  57. Neumann, H. et al. A method for genetically installing site-specific acetylation in recombinant histones defines the effects of H3 K56 acetylation. Mol. Cell. 36, 153–163 (2009). Uses site-specific histone acetylation and single-molecule FRET studies to examine how acetylation of H3K56 can influence the conformation of DNA.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Lammers, M., Neumann, H., Chin, J. W. & James, L. C. Acetylation regulates cyclophilin A catalysis, immunosuppression and HIV isomerization. Nature Chem. Biol. 6, 331–337 (2010). Reports site-specific acetylation of cyclophilin A and structural insights into the interactions between cyclophilin A and cyclosporine, and cyclophilin A and HIV capsid, using X-ray crystallography.

    Article  CAS  Google Scholar 

  59. Arbely, E. et al. Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration. Proc. Natl Acad. Sci. USA 108, 8251–8256 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Oppikofer, M. et al. A dual role of H4K16 acetylation in the establishment of yeast silent chromatin. EMBO J. 30, 2610–2621 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Thao, S., Chen, C.-S., Zhu, H. & Escalante-Semerena, J. C. Nɛ-lysine acetylation of a bacterial transcription factor inhibits its DNA-binding activity. PLoS ONE 5, e15123 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Celic, I. et al. The sirtuins Hst3 and Hst4p preserve genome integrity by controlling histone H3 lysine 56 deacetylation. Curr. Biol. 16, 1280–1289 (2006).

    Article  CAS  PubMed  Google Scholar 

  63. Chen, C.-C. et al. Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair. Cell 134, 231–243 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Das, C., Lucia, M. S., Hansen, K. C. & Tyler, J. K. CBP/p300-mediated acetylation of histone H3 on lysine 56. Nature 459, 113–117 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Driscoll, R., Hudson, A. & Jackson, S. P. Yeast Rtt109 promotes genome stability by acetylating histone H3 on lysine 56. Science 315, 649–652 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Garcia, B. A. et al. Organismal differences in post-translational modifications in histones H3 and H4. J. Biol. Chem. 282, 7641–7655 (2007).

    Article  CAS  PubMed  Google Scholar 

  67. Han, J. et al. Rtt109 acetylates histone H3 Lysine 56 and functions in DNA replication. Science 315, 653–655 (2007).

    Article  CAS  PubMed  Google Scholar 

  68. Hyland, E. M. et al. Insights into the role of histone H3 and histone H4 core modifiable residues in Saccharomyces cerevisiae. Mol. Cell. Biol. 25, 10060–10070 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Li, Q. et al. Acetylation of histone H3 lysine 56 regulates replication-coupled nucleosome assembly. Cell 134, 244–255 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Masumoto, H., Hawke, D., Kobayashi, R. & Verreault, A. A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response. Nature 436, 294–298 (2005).

    Article  CAS  PubMed  Google Scholar 

  71. Ozdemir, A. et al. Characterization of lysine 56 of histone H3 as an acetylation site in Saccharomyces cerevisiae. J. Biol. Chem. 280, 25949–25952 (2005).

    Article  CAS  PubMed  Google Scholar 

  72. Rufiange, A., Jacques, P.-E., Bhat, W., Robert, F. & Nourani, A. Genome-wide replication-independent histone H3 exchange occurs predominantly at promoters and implicates H3 K56 acetylation and Asf1. Mol. Cell 27, 393–405 (2007).

    Article  CAS  PubMed  Google Scholar 

  73. Williams, S. K., Truong, D. & Tyler, J. K. Acetylation in the globular core of histone H3 on lysine-56 promotes chromatin disassembly during transcriptional activation. Proc. Natl Acad. Sci. USA 105, 9000–9005 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Xie, W. et al. Histone H3 lysine 56 acetylation is linked to the core transcriptional network in human embryonic stem cells. Mol. Cell 33, 417–427 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Xu, F., Zhang, K. & Grunstein, M. Acetylation in histone h3 globular domain regulates gene expression in yeast. Cell 121, 375–385 (2005).

    Article  CAS  PubMed  Google Scholar 

  76. Xu, F., Zhang, Q., Zhang, K., Xie, W. & Grunstein, M. Sir2 deacetylates histone H3 lysine 56 to regulate telomeric heterochromatin structure in yeast. Mol. Cell 27, 890–900 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Yang, B., Miller, A. & Kirchmaier, A. L. HST3/HST4-dependent deacetylation of lysine 56 of histone H3 in silent chromatin. Mol. Biol. Cell 19, 4993–5005 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Davey, C. A., Sargent, D. F., Luger, K., Maeder, A. W. & Richmond, T. J. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution. J. Mol. Biol. 319, 1097–1113 (2002).

    Article  CAS  PubMed  Google Scholar 

  79. Koopmans, W. J. A., Buning, R., Schmidt, T. & van Noort, J. spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes. Biophys. J. 97, 195–204 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Roehrl, M. H. A. et al. Selective inhibition of calcineurin–NFAT signaling by blocking protein–protein interaction with small organic molecules. Proc. Natl Acad. Sci. USA 101, 7554–7559 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Franke, E. K., Yuan, H. E. H. & Luban, J. Specific incorporation of cyclophilin A into HIV-1 virions. Nature 372, 359–362 (1994).

    Article  CAS  PubMed  Google Scholar 

  82. Thali, M. et al. Functional association of cyclophilin A with HIV-1 virions. Nature 372, 363–365 (1994).

    Article  CAS  PubMed  Google Scholar 

  83. Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).

    Article  CAS  PubMed  Google Scholar 

  84. Nguyen, D. P., Elliott, T., Holt, M., Muir, T. W. & Chin, J. W. Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. J. Am. Chem. Soc. 133, 11418–11421 (2011).

    Article  CAS  PubMed  Google Scholar 

  85. McGinty, R. K. et al. Structure-activity analysis of semisynthetic nucleosomes: mechanistic insights into the stimulation of Dot1L by ubiquitylated histone H2B. ACS Chem. Biol. 4, 958–968 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Castaneda, C. et al. Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme. J. Am. Chem. Soc. 133, 17855–17868 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Kumar, K. S. A., Spasser, L., Erlich, L. A., Bavikar, S. N. & Brik, A. Total chemical synthesis of di-ubiquitin chains. Angew. Chem. Int. Ed. Engl. 49, 9126–9131 (2010).

    Article  CAS  PubMed  Google Scholar 

  88. Ajish Kumar, K. S., Haj-Yahya, M., Olschewski, D., Lashuel, H. A. & Brik, A. Highly efficient and chemoselective peptide ubiquitylation. Angew. Chem. Int. Ed. Engl. 48, 8090–8094 (2009).

    Article  CAS  PubMed  Google Scholar 

  89. Xu, P. et al. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137, 133–145 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Chatterjee, C., McGinty, R. K., Pellois, J.-P. & Muir, T. W. Auxiliary-mediated site-specific peptide ubiquitylation. Angew. Chem. Int. Ed. Engl. 46, 2814–2818 (2007).

    Article  CAS  PubMed  Google Scholar 

  91. El Oualid, F. et al. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem. Int. Ed. Engl. 49, 10149–10153 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. McGinty, R. K., Kim, J., Chatterjee, C., Roeder, R. G. & Muir, T. W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453, 812–816 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Yang, R., Pasunooti, K. K., Li, F., Liu, X.-W. & Liu, C.-F. Dual native chemical ligation at lysine. J. Am. Chem. Soc. 131, 13592–13593 (2009).

    Article  CAS  PubMed  Google Scholar 

  94. Yang, R. L., Pasunooti, K. K., Li, F. P., Liu, X. W. & Liu, C. F. Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine. Chem. Commun. 46, 7199–7201 (2010).

    Article  CAS  Google Scholar 

  95. Tran, H., Hamada, F., Schwarz-Romond, T. & Bienz, M. Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains. Genes Dev. 22, 528–542 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Deiters, A., Groff, D., Ryu, Y., Xie, J. & Schultz, P. G. A genetically encoded photocaged tyrosine. Angew. Chem. Int. Ed. Engl. 45, 2728–2731 (2006).

    Article  CAS  PubMed  Google Scholar 

  97. Lemke, E. A., Summerer, D., Geierstanger, B. H., Brittain, S. M. & Schultz, P. G. Control of protein phosphorylation with a genetically encoded photocaged amino acid. Nature Chem. Biol. 3, 769–772 (2007).

    Article  CAS  Google Scholar 

  98. Gautier, A., Deiters, A. & Chin, J. W. Light-activated kinases enable temporal dissection of signaling networks in living cells. J. Am. Chem. Soc. 133, 2124–2127 (2011). Uses a photocaged amino acid in MEK to allow specific photoactivation of a subnetwork in MAPK signalling pathway and dissection of key regulatory steps in this pathway.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Chen, P. R. et al. A facile system for encoding unnatural amino acids in mammalian cells. Angew. Chem. Int. Ed. Engl. 48, 4052–4055 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Cohen-Saidon, C., Cohen, A. A., Sigal, A., Liron, Y. & Alon, U. Dynamics and variability of ERK2 response to EGF in individual living cells. Mol. Cell 36, 885–893 (2009).

    Article  CAS  PubMed  Google Scholar 

  101. Burack, W. R. & Sturgill, T. W. The activating dual phosphorylation of MAPK by MEK is nonprocessive. Biochemistry 36, 5929–5933 (1997).

    Article  CAS  PubMed  Google Scholar 

  102. Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

    Article  CAS  PubMed  Google Scholar 

  103. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. & Prasher, D. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    Article  CAS  PubMed  Google Scholar 

  104. Heim, R., Prasher, D. C. & Tsien, R. Y. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl Acad. Sci. USA 91, 12501–12504 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Giepmans, B. N. G., Adams, S. R., Ellisman, M. H. & Tsien, R. Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  CAS  PubMed  Google Scholar 

  106. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905–909 (2005).

    Article  CAS  PubMed  Google Scholar 

  107. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  PubMed  Google Scholar 

  108. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotech. 21, 86–89 (2003).

    Article  CAS  Google Scholar 

  109. Kosaka, N. et al. In vivo stable tumor-specific painting in various colors using dehalogenase-based protein-tag fluorescent ligands. Bioconjug. Chem. 20, 1367–1374 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  PubMed  Google Scholar 

  111. George, N., Pick, H., Vogel, H., Johnsson, N. & Johnsson, K. Specific labeling of cell surface proteins with chemically diverse compounds. J. Am. Chem. Soc. 126, 8896–8897 (2004).

    Article  CAS  PubMed  Google Scholar 

  112. Zhou, Z., Koglin, A., Wang, Y., McMahon, A. P. & Walsh, C. T. An eight residue fragment of an acyl carrier protein suffices for post-translational introduction of fluorescent pantetheinyl arms in protein modification in vitro and in vivo. J. Am. Chem. Soc. 130, 9925–9930 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Yin, J. et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc. Natl Acad. Sci. USA 102, 15815–15820 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Fernandez-Suarez, M. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nature Biotech. 25, 1483–1487 (2007).

    Article  CAS  Google Scholar 

  115. Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl Acad. Sci. USA 107, 10914–10919 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E. & Ploegh, H. L. Sortagging: a versatile method for protein labeling. Nature Chem. Biol. 3, 707–708 (2007).

    Article  CAS  Google Scholar 

  117. Antos, J. M. et al. Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity. J. Am. Chem. Soc. 131, 10800–10801 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Liu, D. S. et al. Diels–Alder cycloaddition for fluorophore targeting to specific proteins inside living cells. J. Am. Chem. Soc. 134, 792–795 (2011).

    Article  CAS  Google Scholar 

  119. Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  PubMed  Google Scholar 

  120. Halo, T. L., Appelbaum, J., Hobert, E. M., Balkin, D. M. & Schepartz, A. Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid. J. Am. Chem. Soc. 131, 438–439 (2008).

    Article  CAS  Google Scholar 

  121. Hinner, M. J. & Johnsson, K. How to obtain labeled proteins and what to do with them. Curr. Opin. Biotechnol. 21, 766–776 (2010).

    Article  CAS  PubMed  Google Scholar 

  122. Xie, J. M. et al. The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination. Nature Biotech. 22, 1297–1301 (2004).

    Article  CAS  Google Scholar 

  123. Walden, H. Selenium incorporation using recombinant techniques. Acta Crystallogr. D Biol. Crystallogr. 66, 352–357 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Cellitti, S. E. et al. In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy. J. Am. Chem. Soc. 130, 9268–9281 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Deiters, A., Geierstanger, B. H. & Schultz, P. G. Site-specific in vivo labeling of proteins for NMR studies. Chembiochem 6, 55–58 (2005).

    Article  CAS  PubMed  Google Scholar 

  126. Hammill, J. T., Miyake-Stoner, S., Hazen, J. L., Jackson, J. C. & Mehl, R. A. Preparation of site-specifically labeled fluorinated proteins for 19F-NMR structural characterization. Nature Protoc. 2, 2601–2607 (2007).

    Article  CAS  Google Scholar 

  127. Jackson, J. C., Hammill, J. T. & Mehl, R. A. Site-specific incorporation of a 19F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity. J. Am. Chem. Soc. 129, 1160–1166 (2007).

    Article  CAS  PubMed  Google Scholar 

  128. Lampe, J. N., Brandman, R., Sivaramakrishnan, S. & de Montellano, P. R. O. Two-dimensional NMR and all-atom molecular dynamics of cytochrome P450 CYP119 reveal hidden conformational substates. J. Biol. Chem. 285, 9594–9603 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Li, C. G. et al. Protein 19F NMR in Escherichia coli. J. Am. Chem. Soc. 132, 321–327 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Wang, L., Brock, A. & Schultz, P. G. Adding L-3-(2-naphthyl)alanine to the genetic code of E. coli. J. Am. Chem. Soc. 124, 1836–1837 (2002).

    Article  CAS  PubMed  Google Scholar 

  131. Lee, H. S., Guo, J., Lemke, E. A., Dimla, R. D. & Schultz, P. G. Genetic incorporation of a small, environmentally sensitive, fluorescent probe into proteins in Saccharomyces cerevisiae. J. Am. Chem. Soc. 131, 12921–12923 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Wang, J., Xie, J. & Schultz, P. G. A genetically encoded fluorescent amino acid. J. Am. Chem. Soc. 128, 8738–8739 (2006).

    Article  CAS  PubMed  Google Scholar 

  133. Summerer, D. et al. A genetically encoded fluorescent amino acid. Proc. Natl Acad. Sci. USA 103, 9785–9789 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Chapman, E., Farr, G. W., Furtak, K. & Horwich, A. L. A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL. Bioorg. Med. Chem. Lett. 19, 811–813 (2009).

    Article  CAS  PubMed  Google Scholar 

  135. Schultz, K. C. et al. A genetically encoded infrared probe. J. Am. Chem. Soc. 128, 13984–13985 (2006).

    Article  CAS  PubMed  Google Scholar 

  136. Charbon, G. et al. Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid. Bioorg. Med. Chem. Lett. 21, 6067–6070 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Ye, S. X., Huber, T., Vogel, R. & Sakmar, T. P. FTIR analysis of GPCR activation using azido probes. Nature Chem. Biol. 5, 397–399 (2009).

    Article  CAS  Google Scholar 

  138. Ye, S. X. et al. Tracking G-protein-coupled receptor activation using genetically encoded infrared probes. Nature 464, 1386–1389 (2010). Describes the use of infrared probes to follow the conformational changes that occur in structural intermediates of the GPCR rhodopsin during activation in response to light.

    Article  CAS  PubMed  Google Scholar 

  139. Neumann, H., Slusarczyk, A. L. & Chin, J. W. De novo generation of mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs. J. Am. Chem. Soc. 132, 2142–2144 (2010).

    Article  CAS  PubMed  Google Scholar 

  140. Neumann, H., Wang, K., Davis, L., Garcia-Alai, M. & Chin, J. W. Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. Nature 464, 441–444 (2010). References 139 and 140 detail approaches for encoding more than one unnatural amino acid through engineering the ribosome and generating new synthetase tRNA pairs.

    Article  CAS  PubMed  Google Scholar 

  141. Muralidharan, V. & Muir, T. W. Protein ligation: an enabling technology for the biophysical analysis of proteins. Nature Methods 3, 429–438 (2006).

    Article  CAS  PubMed  Google Scholar 

  142. Vila-Perelló, M. & Muir, T. W. Biological applications of protein splicing. Cell 143, 191–200 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  143. Boyce, M. & Bertozzi, C. R. Bringing chemistry to life. Nature Methods 8, 638–642 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Zhang, Z. et al. A new strategy for the site-specific modification of proteins in vivo. Biochemistry 42, 6735–6746 (2003).

    Article  CAS  PubMed  Google Scholar 

  145. Ye, S. et al. Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. J. Biol. Chem. 283, 1525–1533 (2008).

    Article  CAS  PubMed  Google Scholar 

  146. Tsao, M. L., Tian, F. & Schultz, P. G. Selective Staudinger modification of proteins containing p-azidophenylalanine. Chembiochem 6, 2147–2149 (2005).

    Article  CAS  PubMed  Google Scholar 

  147. Tian, F., Tsao, M.-L. & Schultz, P. G. A phage display system with unnatural amino acids. J. Am. Chem. Soc. 126, 15962–15963 (2004).

    Article  CAS  PubMed  Google Scholar 

  148. Brustad, E. M., Lemke, E. A., Schultz, P. G. & Deniz, A. A. A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer. J. Am. Chem. Soc. 130, 17664–17665 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Agard, N. J., Prescher, J. A. & Bertozzi, C. R. A strain-promoted 3 + 2 azide-alkyne cycloaddition for covalent modification of blomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

    Article  CAS  PubMed  Google Scholar 

  150. Yanagisawa, T. et al. Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(varepsilon)-(o-Azidobenzyloxycarbonyl) lysine for site-specific protein modification. Chem. Biol. 15, 1187–1197 (2008).

    Article  CAS  PubMed  Google Scholar 

  151. Wang, J. et al. A biosynthetic route to photoclick chemistry on proteins. J. Am. Chem. Soc. 132, 14812–14818 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Wang, Y., Song, W., Hu, W. J. & Lin, Q. Fast alkene functionalization in vivo by photoclick chemistry: HOMO lifting of nitrile imine dipoles. Angew. Chem. Int. Ed. Engl. 48, 5330–5333 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Song, W., Wang, Y., Qu, J. & Lin, Q. Selective functionalization of a genetically encoded alkene-containing protein via “photoclick chemistry” in bacterial cells. J. Am. Chem. Soc. 130, 9654–9655 (2008).

    Article  CAS  PubMed  Google Scholar 

  154. Song, W., Wang, Y., Qu, J., Madden, M. M. & Lin, Q. A photoinducible 1,3-dipolar cycloaddition reaction for rapid, selective modification of tetrazole-containing proteins. Angew. Chem. Int. Ed. Engl. 47, 2832–2835 (2008).

    Article  CAS  PubMed  Google Scholar 

  155. Nguyen, D. P. et al. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNACUA pair and click chemistry. J. Am. Chem. Soc. 131, 8720–8721 (2009).

    Article  CAS  PubMed  Google Scholar 

  156. Fekner, T., Li, X., Lee, M. M. & Chan, M. K. A pyrrolysine analogue for protein click chemistry. Angew. Chem. Int. Ed. Engl. 48, 1633–1635 (2009).

    Article  CAS  PubMed  Google Scholar 

  157. Deiters, A., Cropp, T. A., Summerer, D., Mukherji, M. & Schultz, P. G. Site-specific PEGylation of proteins containing unnatural amino acids. Bioorg. Med. Chem. Lett. 14, 5743–5745 (2004).

    Article  CAS  PubMed  Google Scholar 

  158. Carrico, Z. M., Romanini, D. W., Mehl, R. A. & Francis, M. B. Oxidative coupling of peptides to a virus capsid containing unnatural amino acids. Chem. Commun. (Camb.) 1205–1207 (2008).

  159. Wang, L., Zhang, Z., Brock, A. & Schultz, P. G. Addition of the keto functional group to the genetic code of Escherichiacoli. Proc. Natl Acad. Sci. USA 100, 56–61 (2003).

    Article  CAS  PubMed  Google Scholar 

  160. Mehl, R. A. et al. Generation of a bacterium with a 21 amino acid genetic code. J. Am. Chem. Soc. 125, 935–939 (2003).

    Article  CAS  PubMed  Google Scholar 

  161. Fleissner, M. R. et al. Site-directed spin labeling of a genetically encoded unnatural amino acid. Proc. Natl Acad. Sci. USA 106, 21637–21642 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  162. Zeng, H., Xie, J. & Schultz, P. G. Genetic introduction of a diketone-containing amino acid into proteins. Bioorg. Med. Chem. Lett. 16, 5356–5359 (2006).

    Article  CAS  PubMed  Google Scholar 

  163. Huang, Y. et al. Genetic incorporation of an aliphatic keto-containing amino acid into proteins for their site-specific modifications. Bioorg. Med. Chem. Lett. 20, 878–880 (2010).

    Article  CAS  PubMed  Google Scholar 

  164. Deiters, A. et al. Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. J. Am. Chem. Soc. 125, 11782–11783 (2003).

    Article  CAS  PubMed  Google Scholar 

  165. Deiters, A. & Schultz, P. G. In vivo incorporation of an alkyne into proteins in Escherichia coli. Bioorg. Med. Chem. Lett. 15, 1521–1524 (2005).

    Article  CAS  PubMed  Google Scholar 

  166. Plass, T., Milles, S., Koehler, C., Schultz, C. & Lemke, E. A. Genetically encoded copper-free click chemistry. Angew. Chem. Int. Ed. Engl. 50, 3878–3881 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Tong, G. J., Hsiao, S. C., Carrico, Z. M. & Francis, M. B. Viral capsid DNA aptamer conjugates as multivalent cell-targeting vehicles. J. Am. Chem. Soc. 131, 11174–11178 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. Kodama, K. et al. Regioselective carbon–carbon bond formation in proteins with palladium catalysis; new protein chemistry by organometallic chemistry. Chembiochem 7, 134–139 (2006).

    Article  CAS  PubMed  Google Scholar 

  169. Kodama, K. et al. Site-specific functionalization of proteins by organopalladium reactions. Chembiochem 8, 232–238 (2007).

    Article  CAS  PubMed  Google Scholar 

  170. Spicer, C. D. & Davis, B. G. Palladium-mediated site-selective Suzuki–Miyaura protein modification at genetically encoded aryl halides. Chem. Commun. 47, 1698–1700 (2011).

    Article  CAS  Google Scholar 

  171. Brustad, E. et al. A genetically encoded boronate-containing amino acid. Angew. Chem. Int. Ed. Engl. 47, 8220–8223 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  172. Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction Nature Chem. 5 Feb 2012 (doi:10.1038/nchem.1250). Describes rapid and site-specific fluorescent labelling of a protein on mammalian cells through genetic code expansion.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. Agard, N. J., Baskin, J. M., Prescher, J. A., Lo, A. & Bertozzi, C. R. A comparative study of bioorthogonal reactions with azides. ACS Chem. Biol. 1, 644–648 (2006).

    Article  CAS  PubMed  Google Scholar 

  174. Devaraj, N. K., Weissleder, R. & Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug. Chem. 19, 2297–2299 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  175. Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  176. Hengming, K. Similarities and differences between human cyclophilin A and other β-barrel structures: structural refinement at 1.63 Å resolution. J. Mol. Biol. 228, 539–550 (1992).

    Article  Google Scholar 

  177. Cook, W. J., Jeffrey, L. C., Carson, M., Chen, Z. & Pickart, C. M. Structure of a diubiquitin conjugate and a model for interaction with ubiquitin conjugating enzyme (E2). J. Biol. Chem. 267, 16467–16471 (1992).

    Article  CAS  PubMed  Google Scholar 

  178. Komander, D. et al. Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains. EMBO Rep. 10, 466–473 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  179. Bremm, A., Freund, S. M. V. & Komander, D. Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne. Nature Struct. Mol. Biol. 17, 939–947 (2010).

    Article  CAS  Google Scholar 

  180. Ohren, J. F. et al. Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition. Nature Struct. Mol. Biol. 11, 1192–1197 (2004).

    Article  CAS  Google Scholar 

  181. Li, J., Edwards, P. C., Burghammer, M., Villa, C. & Schertler, G. F. X. Structure of bovine rhodopsin in a trigonal crystal form. J. Mol. Biol. 343, 1409–1438 (2004).

    Article  CAS  PubMed  Google Scholar 

  182. Scheerer, P. et al. Crystal structure of opsin in its G-protein-interacting conformation. Nature 455, 497–502 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to the UK Medical Research Council for funding(U105181009 and MC_UP_A024_1008) and to K. Lang and S. Hancock for help with the manuscript.

Author information

Authors and Affiliations

  1. Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK

    Lloyd Davis & Jason W. Chin

Authors
  1. Lloyd Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jason W. Chin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jason W. Chin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Related links

Related links

DATABASES

Protein Databank

1AAR

1KX5

1GZM

1S9J

2CPL

2JF5

2W9N

2XEW

2XK5

3DIN

3DQB

FURTHER INFORMATION

Jason W. Chin's homepage

Glossary

Selective pressure incorporation

Bacteria that are auxotrophic for a natural amino acid are used in conjunction with a related unnatural amino acid, leading to the incorporation of the unnatural amino acid throughout the cell's proteome.

TAP tagging

(Tandem affinity purification tagging). A process in which a protein is carboxy-terminally tagged with a peptide containing a calmodulin-binding peptide, a TEV protease cleavage site and protein A. The protein is first purified using Immunoglobulin G-coated beads that bind protein A. The protein fusion is then cleaved from the gene of interest by the TEV protease and purified.

Chaperones

Proteins that assist other macromolecules in folding and/or unfolding and assembly and/or disassembly.

Elongation factor-Tu

(EF-Tu). A protein that binds to aminoacylated tRNAs and delivers them to the ribosome for protein synthesis.

Nucleosome

A unit of DNA packaging in eukaryotes in which a length of DNA is wrapped around an octamer of histone proteins.

SWI/SNF

(Switch/sucrose nonfermentable). An ATP-dependent multiprotein nucleosome-remodelling complex that is found in yeast.

RSC

(Remodels the structure of chromatin). An ATP-dependent multiprotein nucleosome-remodelling complex.

Prolyl isomerase

An enzyme that is responsible for the cis–trans isomerization of peptide bonds that are on the amino-terminal side of Pro residues.

Enthalpy

A measure (in thermodynamics) of the total internal energy of a system plus its pressure multiplied by its volume.

Entropy

A thermodynamic property that can be used to determine the energy that is not available for work.

E1, E2 and E3

The enzymes by which ubiquitin is added to cellular proteins. The E1 activates ubiquitin by attaching the molecule to its active site Cys residue. The E2 then binds the ubiquitin molecule, also by a Cys residue. The E2 then binds an E3 ligase which binds the target protein and catalyses the transfer of the ubiquitin to a Lys residue of the target protein.

Atypical ubiquitin chains

Ubiquitin chains that are not linked by the common Lys48 or Lys63 linkages but by one of the other Lys residues of ubiquitin.

Sumoylation

The covalent attachment of small ubiquitin-like modifier (SUMO) to a substrate protein.

Neddylation

A process, analogous to ubiquitylation, in which ubiquitin-like protein NEDD8 is conjugated to a protein substrate.

Distributive

A mode of activation in which two or more events occur independently. In the case of ERK2, which is diphosphorylated when activated, the MEK1–ERK2 complex dissociates after each phosphorylation event.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Davis, L., Chin, J. Designer proteins: applications of genetic code expansion in cell biology. Nat Rev Mol Cell Biol 13, 168–182 (2012). https://doi.org/10.1038/nrm3286

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrm3286

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing