Skip to main content

Thank you for visiting 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.

Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET

A Corrigendum to this article was published on 23 January 2014

This article has been updated


The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Optimizing Pyl tRNA(N8)XXXX for incorporating unnatural amino acids in response to quadruplet codons decoded by ribo-Q1.
Figure 2: Evolved Pyl tRNA(N8)XXXX direct the efficient unnatural amino acid incorporation in response to quadruplet codons decoded by ribo-Q1.
Figure 3: Efficient incorporation of multiple distinct unnatural amino acids into a single polypeptide.
Figure 4: Efficient incorporation of a matrix of pairs of unnatural amino acids, including photocrosslinkers and chemical handles (azides, alkenes, alkynes and tetrazines), demonstrates generality.
Figure 5: 5 and 8 do not react with each other in a protein, but can be labelled efficiently with 10 and 9.
Figure 6: Site-specific double-labelling of CaM with a FRET pair to follow changes in protein conformation.

Change history

  • 07 January 2014

    In the version of this Article originally published, the middle initial of the co-author Nabil M. Wilf was incorrect, and the Acknowledgements section was missing a reference to the European Research Council; the statement should have read: "We thank the Medical Research Council (U105181009, UD99999908) and the European Research Council (MC-A024-PG0A) for financial support. We thank S-P. Chew (MRC-LMB Mass Spectrometry) for obtaining MALDI data." These errors have now been corrected in the online versions of the Article.


  1. 1

    Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Cell Biol. 3, 906–918 (2002).

    CAS  Google Scholar 

  2. 2

    Kajihara, D. et al. FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nature Methods 3, 923–929 (2006).

    CAS  PubMed  Google Scholar 

  3. 3

    Scott, C.P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl Acad. Sci. USA 96, 13638–13643 (1999).

    CAS  PubMed  Google Scholar 

  4. 4

    Li, P. & Roller, P. P. Cyclization strategies in peptide derived drug design. Curr. Top. Med. Chem. 2, 325–341 (2002).

    CAS  PubMed  Google Scholar 

  5. 5

    Wang, K., Schmied, W. H. & Chin, J. W. Reprogramming the genetic code: from triplet to quadruplet codes. Angew. Chem. Int. Ed. 51, 2288–2297 (2012).

    CAS  Google Scholar 

  6. 6

    Davis, L. & Chin, J. W. Designer proteins: applications of genetic code expansion in cell biology. Nature Rev. Mol. Cell Biol. 13, 168–182 (2012).

    CAS  Google Scholar 

  7. 7

    Chin, J. W. Molecular biology. Reprogramming the genetic code. Science 336, 428–429 (2012).

    CAS  PubMed  Google Scholar 

  8. 8

    Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. (2014).

    Google Scholar 

  9. 9

    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).

    CAS  PubMed  Google Scholar 

  10. 10

    Rackham, O. & Chin, J. W. A network of orthogonal ribosome X mRNA pairs. Nature Chem. Biol. 1, 159–166 (2005).

    CAS  Google Scholar 

  11. 11

    Wang, K., Neumann, H., Peak-Chew, S. Y. & Chin, J. W. Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Nature Biotechnol. 25, 770–777 (2007).

    Google Scholar 

  12. 12

    An, W. & Chin, J. W. Synthesis of orthogonal transcription–translation networks. Proc. Natl Acad. Sci. USA 106, 8477–8482 (2009).

    CAS  PubMed  Google Scholar 

  13. 13

    Atkins, J. F. & Bjork, G. R. A gripping tale of ribosomal frameshifting: extragenic suppressors of frameshift mutations spotlight P-site realignment. Microbiol. Mol. Biol. Rev. 73, 178–210 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Stahl, G., McCarty, G. P. & Farabaugh, P. J. Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding. Trends Biochem. Sci. 27, 178–183 (2002).

    CAS  PubMed  Google Scholar 

  15. 15

    Wan, W. et al. A facile system for genetic incorporation of two different noncanonical amino acids into one protein in Escherichia coli. Angew. Chem. Int. Ed. 49, 3211–3214 (2010).

    CAS  Google Scholar 

  16. 16

    Wu, B., Wang, Z., Huang, Y. & Liu, W. R. Catalyst-free and site-specific one-pot dual-labeling of a protein directed by two genetically incorporated noncanonical amino acids. ChemBioChem 13, 1405–1408 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Chatterjee, A., Sun, S. B., Furman, J. L., Xiao, H. & Schultz, P. G. A versatile platform for single- and multiple-unnatural amino acid mutagenesis in Escherichia coli. Biochemistry 52, 1828–1837 (2013).

    CAS  PubMed  Google Scholar 

  18. 18

    Ambrogelly, A. et al. Pyrrolysine is not hardwired for cotranslational insertion at UAG codons. Proc. Natl Acad. Sci. USA 104, 3141–3146 (2007).

    CAS  PubMed  Google Scholar 

  19. 19

    Jiang, R. & Krzycki, J. A. PylSn and the homologous N-terminal domain of pyrrolysyl-tRNA synthetase bind the tRNA that is essential for the genetic encoding of pyrrolysine. J. Biol. Chem. 287, 32738–32746 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Magliery, T. J., Anderson, J. C. & Schultz, P. G. Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of ‘shifty’ four-base codons with a library approach in Escherichia coli. J. Mol. Biol. 307, 755–769 (2001).

    CAS  PubMed  Google Scholar 

  21. 21

    Niu, W., Schultz, P. G. & Guo, J. An expanded genetic code in mammalian cells with a functional quadruplet codon. ACS Chem. Biol. 8, 1640–1645 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Beuning, P. J. & Musier-Forsyth, K. Transfer RNA recognition by aminoacyl-tRNA synthetases. Biopolymers 52, 1–28 (1999).

    CAS  PubMed  Google Scholar 

  23. 23

    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).

    CAS  PubMed  Google Scholar 

  24. 24

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

    CAS  PubMed  Google Scholar 

  25. 25

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

    CAS  PubMed  Google Scholar 

  26. 26

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

    CAS  PubMed  Google Scholar 

  27. 27

    Seitchik, J. L. et al. Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes. J. Am. Chem. Soc. 134, 2898–2901 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    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. 99, 11020–11024 (2002).

    CAS  PubMed  Google Scholar 

  29. 29

    Sasmal, P. K. et al. Catalytic azide reduction in biological environments. ChemBioChem 13, 1116–1120 (2012).

    CAS  PubMed  Google Scholar 

  30. 30

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Devaraj, N. K. & Weissleder, R. Biomedical applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816–827 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. (2014).

  33. 33

    Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nature Chem. 4, 298–304 (2012).

    CAS  Google Scholar 

  34. 34

    Lang, K. et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J. Am. Chem. Soc. 134, 10317–10320 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Plass, T. et al. Amino acids for Diels–Alder reactions in living cells. Angew. Chem. Int. Ed. 51, 4166–4170 (2012).

    CAS  Google Scholar 

  36. 36

    Borrmann, A. et al. Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation. ChemBioChem 13, 2094–2099 (2012).

    CAS  PubMed  Google Scholar 

  37. 37

    Balcar, J., Chrisam, G., Huber, F. X. & Sauer, J. Reaktivitaet von Stickstoff-Heterocyclen gegenueber Cyclooctin als Dienophil. Tetrahedron Lett. 24, 1481–1484 (1983).

    CAS  Google Scholar 

  38. 38

    Thalhammer, F., Wallfahrer, U. & Sauer, J. Reaktivitaet einfacher offenkettiger und cyclischer Dienophile bei Diels–Alder Reaktionen mit inversem Elektronenbedarf. Tetrahedron Lett. 31, 6851–6854 (1990).

    CAS  Google Scholar 

  39. 39

    Kaya, E. et al. A Genetically encoded norbornene amino acid for the mild and selective modification of proteins in a copper-free click reaction. Angew. Chem. Int. Ed. 51, 4466–4469 (2012).

    CAS  Google Scholar 

  40. 40

    Lang, K. & Chin, J. W. Bioorthogonal reactions for labeling proteins. ACS Chem. Biol. 9, 16–20 (2014).

    CAS  PubMed  Google Scholar 

  41. 41

    Hellstrand, E. et al. Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter-domain electrostatic repulsion. FEBS J 280, 2675–2687 (2013).

    CAS  PubMed  Google Scholar 

  42. 42

    Wissner, R. F., Batjargal, S., Fadzen, C. M. & Petersson, E. J. Labeling proteins with fluorophore/thioamide Förster resonant energy transfer pairs by combining unnatural amino acid mutagenesis and native chemical ligation. J. Am. Chem. Soc. 135, 6529–6540 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Hoeflich, K. P. & Ikura, M. Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108, 739–742 (2002).

    CAS  PubMed  Google Scholar 

  44. 44

    Carafoli, E. Calcium signaling: a tale for all seasons. Proc. Natl Acad. Sci. USA 99, 1115–1122 (2002).

    CAS  PubMed  Google Scholar 

  45. 45

    Taylor, D. A., Sack, J. S., Maune, J. F., Beckingham, K. & Quiocho, F. A. Structure of a recombinant calmodulin from Drosophila melanogaster refined at 2.2-A resolution. J. Biol. Chem. 266, 21375–21380 (1991).

    CAS  PubMed  Google Scholar 

  46. 46

    Fallon, J. L. & Quiocho, F. A. A closed compact structure of native Ca2+-calmodulin. Structure 11, 1303–1307 (2003).

    CAS  PubMed  Google Scholar 

  47. 47

    Linse, S., Helmersson, A. & Forsen, S. Calcium binding to calmodulin and its globular domains. J. Biol. Chem. 266, 8050–8054 (1991).

    CAS  PubMed  Google Scholar 

  48. 48

    Babu, Y. S. et al. Three-dimensional structure of calmodulin. Nature 315, 37–40 (1985).

    CAS  PubMed  Google Scholar 

  49. 49

    Chou, J. J., Li, S., Klee, C. B. & Bax, A. Solution structure of Ca2+-calmodulin reveals flexible hand-like properties of its domains. Nature Struct. Biol. 8, 990–997 (2001).

    CAS  PubMed  Google Scholar 

  50. 50

    Johnson, C. K. Calmodulin, conformational states, and calcium signaling. A single-molecule perspective. Biochemistry 45, 14233–14246 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Wu, G., Gao, Z., Dong, A. & Yu, S. Calcium-induced changes in calmodulin structural dynamics and thermodynamics. Int. J. Biol. Macromol. 50, 1011–1017 (2012).

    CAS  PubMed  Google Scholar 

  52. 52

    Porumb, T. Determination of calcium-binding constants by flow dialysis. Anal. Biochem. 220, 227–237 (1994).

    CAS  PubMed  Google Scholar 

  53. 53

    Haiech, J., Klee, C. B. & Demaille, J. G. Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Biochemistry 20, 3890–3897 (1981).

    CAS  PubMed  Google Scholar 

  54. 54

    Stefan, M. I., Edelstein, S. J. & Le Novere, N. An allosteric model of calmodulin explains differential activation of PP2B and CaMKII. Proc. Natl Acad. Sci. USA 105, 10768–10773 (2008).

    CAS  PubMed  Google Scholar 

Download references


We thank the Medical Research Council (U105181009, UD99999908) and the European Research Council (MC-A024-5PG0A) for financial support. We thank S-P. Chew (MRC-LMB Mass Spectrometry) for obtaining MALDI data.

Author information




J.W.C. and K.W. conceived the project. J.W.C., K.W. and A.S. planned and designed experiments and wrote the manuscript, with input from the other authors. R.A.M. provided MjTetPheRS and compound 5 for pilot experiments. All other authors performed experiments or provided reagents. K.W. and A.S. contributed equally to this work.

Corresponding author

Correspondence to Jason W. Chin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 18909 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, K., Sachdeva, A., Cox, D. et al. Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET. Nature Chem 6, 393–403 (2014).

Download citation

Further reading


Quick links