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

Journal name:
Nature Chemistry
Volume:
6,
Pages:
393–403
Year published:
DOI:
doi:10.1038/nchem.1919
Received
Accepted
Published online
Corrected online

Abstract

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.

At a glance

Figures

  1. Optimizing Pyl tRNA(N8)XXXX for incorporating unnatural amino acids in response to quadruplet codons decoded by ribo-Q1.
    Figure 1: Optimizing Pyl tRNA(N8)XXXX for incorporating unnatural amino acids in response to quadruplet codons decoded by ribo-Q1.

    a, Nucleotides that are targeted for mutagenesis in the anticodon stem loop of Pyl tRNA(N8)XXXX are represented in orange on tRNA bound to the ribosome, and the rest of the tRNA is in yellow. rRNA is in pale green with the ribo-Q1 mutation sites in red, and mRNA in purple. The structural image is based on PDB ID 2J00, created with Pymol (www.pymol.org). b, The anticodon stem loop of Pyl tRNA(N8)XXXX. The nucleotides in orange are randomized in each library; codon sequences and mRNAs are in purple and anticodons in grey. c, Two-step selection procedure to identify specific and efficient Pyl tRNA(N8)XXXX library members in orthogonal translation. A negative O-barnase selection is followed by a positive O-cat selection. Negative selection in the absence of unnatural amino acids eliminates Pyl tRNA(N8)XXXX library members that are mis-aminoacylated with natural amino acids by endogenous aminoacyl synthetases. Subsequent positive selection enriches evolved Pyl tRNA(N8)XXXX library members that are aminoacylated with the added unnatural amino acid by ​PylRS and decoded efficiently at quadruplet codons by ribo-Q1. a.a., amino acid; cat, chloramphenicol acetyl transferase; O-mRNA, orthogonal mRNA.

  2. Evolved Pyl tRNA(N8)XXXX direct the efficient unnatural amino acid incorporation 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.

    a, The selected anticodon stem-loop sequences of evolved Pyl tRNAXXXX, and the corresponding transplant sequences. The anticodons are in grey, and the nucleotides mutated in the library are shown in colour. Positions where the parental sequence is selected are in orange, and positions where new nucleotides are selected are in red. b, Evolved Pyl tRNAXXXX enhance substantially the incorporation of unnatural amino acids by ribo-Q1 in response to quadruplet codons when compared to the corresponding transplant Pyl tRNAXXXX. The unnatural amino acid-dependent decoding of quadruplet codons in the O-cat111XXXX was measured by survival on increasing concentrations of ​chloramphenicol (​Cm). c, Diverse unnatural amino acids are incorporated efficiently in recombinant proteins in response to quadruplet codons using ​PylRS/Pyl tRNAXXXX with orthogonal translation. Full gels are given in Supplementary Fig. 4.

  3. Efficient incorporation of multiple distinct unnatural amino acids into a single polypeptide.
    Figure 3: Efficient incorporation of multiple distinct unnatural amino acids into a single polypeptide.

    a, Site-specific incorporation of 1 and 4. AGGA replaces the first codon and UAG replaces the 40th codon in the cam open reading frame of O-gst-​cam to make O-gst-​cam1AGGA+40TAG. Decoding of both the AGGA and TAG codons by ribo-Q1 produces full-length Gst-​CaM, and failure to decode these codons leads to premature termination of the polypeptide. b,c, The site-specific incorporation efficiency of 1 and 4 is improved by reducing the number of plasmids. d, The newly evolved ​PylRS/tRNAUACU pair substantially increases the efficiency of double incorporation. e, Incorporating two distinct unnatural amino acids using two distinct quadruplet codons. Full gels are given in Supplementary Fig. 7.

  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 4: Efficient incorporation of a matrix of pairs of unnatural amino acids, including photocrosslinkers and chemical handles (azides, alkenes, alkynes and tetrazines), demonstrates generality.

    Cells contained O-gst-​cam1TAG+40AGTA and ribo-Q1 expressed from an RSF plasmid, the pSUP MjAzPheRS/tRNACUA plasmid (or a variant specific for the relevant substrate) and the pCDF ​PylRS/evolved tRNAUACU plasmid. All combinations of ​PylRS substrates (13) and ​MjTyrRS active-site variant substrates (47) were incorporated in a 3 × 4 matrix. We further confirmed the incorporation of distinct unnatural amino acids by ESI-MS and MALDI-MS (Supplementary Fig. 8). Full gels are given in Supplementary Fig. 8. We observed an additional peak for protein samples with 4 that corresponds to the reduction of the azide to an amine.

  5. 5 and 8 do not react with each other in a protein, but can be labelled efficiently with 10 and 9.
    Figure 5: 5 and 8 do not react with each other in a protein, but can be labelled efficiently with 10 and 9.

    a, 5 and 8 do not react when placed in proximity within a protein. ​CaM518149 was purified from cells that bear pRSF ribo-Q1 O-gst-​cam1TAG+149AGTA, pSUP MjTetPheRS/tRNACUA and pCDF NorKRS×3/evolved tRNAUACU. ​CaM412149 undergoes a Cu(I)-catalysed click reaction to cyclize the protein (right gel panel). ​CaM518149 does not cyclize, as judged by mobility shift (compare left and right panels) and ESI-MS (Supplementary Fig. 10). b, Rate constants for the indicated reactions. c, ​CaM51 and ​CaM840 were incubated with 100 molar equiv. 10 at 25 °C. Only ​CaM51 was labelled with 10, which yielded ​CaM5-101. Labelling was visualized using a Typhoon Imager and the resulting time-dependent fluorescence was used to calculate the on-protein labelling rate constant. All measurements were repeated twice and the error bars represent the standard deviation. ESI-MS confirmed that protein labelling was quantitative. d, ​CaM51 and ​CaM840 were incubated with 100 molar equiv. 9 at 25 °C. Only ​CaM840 was labelled with 9, which yielded ​CaM8-940. The labelling reaction was analysed as described in c. ESI-MS confirmed that labelling was quantitative.

  6. Site-specific double-labelling of CaM with a FRET pair to follow changes in protein conformation.
    Figure 6: Site-specific double-labelling of ​CaM with a FRET pair to follow changes in protein conformation.

    a, Strategy for protein double-labelling via inverse electron-demand Diels–Alder reactions. b, Quantitative and site-specific double-labelling of ​CaM51840. Purified ​CaM51840 (lane 1) was labelled with 100 equiv. (200 µM) 10, which yielded ​CaM5-101840 (lane 2), or 100 equiv. 9, which yielded ​CaM518-940 (lane 3). ​CaM518-940 was labelled with 100 equiv. 10, which yielded ​CaM5-1018-940 (lane 4). Labelling was visualized by fluorescence imaging and led to a mobility shift. All labelling reactions were quantitative, as confirmed by ESI-MS. ​CaM51840 (blue peak, calculated mass = 18,081 Da, observed mass = 18,079 Da), ​CaM518-940 (green peak, calculated mass = 18,635, observed mass = 18,632), ​CaM5-1018-940 (orange peak, calculated mass = 19,336, observed mass = 19,330). c, Fluorescence spectra of ​CaM5-1018-9149 (following donor excitation at 485 nm) in the presence of increasing concentrations of ​urea. d, The relative donor-fluorescence intensity from doubly labelled ​CaM5-1018-940 as a function of Ca2+ concentration. All measurements were repeated at least six times and the error bars represent the standard deviation. K1 and K2 are for the observed transitions; R2 = 0.9005; KD1, KD2, KD3 and KD4 are the reported KD (dissociation constants) values for sequential Ca2+ binding51.

Change history

Corrected online 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.

References

  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, 906918 (2002).
  2. Kajihara, D. et al. FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids. Nature Methods 3, 923929 (2006).
  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, 1363813643 (1999).
  4. Li, P. & Roller, P. P. Cyclization strategies in peptide derived drug design. Curr. Top. Med. Chem. 2, 325341 (2002).
  5. Wang, K., Schmied, W. H. & Chin, J. W. Reprogramming the genetic code: from triplet to quadruplet codes. Angew. Chem. Int. Ed. 51, 22882297 (2012).
  6. Davis, L. & Chin, J. W. Designer proteins: applications of genetic code expansion in cell biology. Nature Rev. Mol. Cell Biol. 13, 168182 (2012).
  7. Chin, J. W. Molecular biology. Reprogramming the genetic code. Science 336, 428429 (2012).
  8. Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. http://dx.doi.org/10.1146/annurev-biochem-060713-035737 (2014).
  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, 441444 (2010).
  10. Rackham, O. & Chin, J. W. A network of orthogonal ribosome X mRNA pairs. Nature Chem. Biol. 1, 159166 (2005).
  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, 770777 (2007).
  12. An, W. & Chin, J. W. Synthesis of orthogonal transcription–translation networks. Proc. Natl Acad. Sci. USA 106, 84778482 (2009).
  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).
  14. Stahl, G., McCarty, G. P. & Farabaugh, P. J. Ribosome structure: revisiting the connection between translational accuracy and unconventional decoding. Trends Biochem. Sci. 27, 178183 (2002).
  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, 32113214 (2010).
  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, 14051408 (2012).
  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, 18281837 (2013).
  18. Ambrogelly, A. et al. Pyrrolysine is not hardwired for cotranslational insertion at UAG codons. Proc. Natl Acad. Sci. USA 104, 31413146 (2007).
  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, 3273832746 (2012).
  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, 755769 (2001).
  21. Niu, W., Schultz, P. G. & Guo, J. An expanded genetic code in mammalian cells with a functional quadruplet codon. ACS Chem. Biol. 8, 16401645 (2013).
  22. Beuning, P. J. & Musier-Forsyth, K. Transfer RNA recognition by aminoacyl-tRNA synthetases. Biopolymers 52, 128 (1999).
  23. Chin, J. W. et al. Addition of p-azido-l-phenylalanine to the genetic code of Escherichia coli. J. Am. Chem. Soc. 124, 90269027 (2002).
  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, 11871197 (2008).
  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, 87208721 (2009).
  26. Deiters, A. & Schultz, P. G. In vivo incorporation of an alkyne into proteins in Escherichia coli. Bioorg. Med. Chem. Lett. 15, 15211524 (2005).
  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, 28982901 (2012).
  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, 1102011024 (2002).
  29. Sasmal, P. K. et al. Catalytic azide reduction in biological environments. ChemBioChem 13, 11161120 (2012).
  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, 1351813519 (2008).
  31. Devaraj, N. K. & Weissleder, R. Biomedical applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816827 (2011).
  32. Lang, K. & Chin, J. W. Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. http://dx.doi.org/10.1021/cr400355w (2014).
  33. Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nature Chem. 4, 298304 (2012).
  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, 1031710320 (2012).
  35. Plass, T. et al. Amino acids for Diels–Alder reactions in living cells. Angew. Chem. Int. Ed. 51, 41664170 (2012).
  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, 20942099 (2012).
  37. Balcar, J., Chrisam, G., Huber, F. X. & Sauer, J. Reaktivitaet von Stickstoff-Heterocyclen gegenueber Cyclooctin als Dienophil. Tetrahedron Lett. 24, 14811484 (1983).
  38. Thalhammer, F., Wallfahrer, U. & Sauer, J. Reaktivitaet einfacher offenkettiger und cyclischer Dienophile bei Diels–Alder Reaktionen mit inversem Elektronenbedarf. Tetrahedron Lett. 31, 68516854 (1990).
  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, 44664469 (2012).
  40. Lang, K. & Chin, J. W. Bioorthogonal reactions for labeling proteins. ACS Chem. Biol. 9, 1620 (2014).
  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, 26752687 (2013).
  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, 65296540 (2013).
  43. Hoeflich, K. P. & Ikura, M. Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108, 739742 (2002).
  44. Carafoli, E. Calcium signaling: a tale for all seasons. Proc. Natl Acad. Sci. USA 99, 11151122 (2002).
  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, 2137521380 (1991).
  46. Fallon, J. L. & Quiocho, F. A. A closed compact structure of native Ca2+-calmodulin. Structure 11, 13031307 (2003).
  47. Linse, S., Helmersson, A. & Forsen, S. Calcium binding to calmodulin and its globular domains. J. Biol. Chem. 266, 80508054 (1991).
  48. Babu, Y. S. et al. Three-dimensional structure of calmodulin. Nature 315, 3740 (1985).
  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, 990997 (2001).
  50. Johnson, C. K. Calmodulin, conformational states, and calcium signaling. A single-molecule perspective. Biochemistry 45, 1423314246 (2006).
  51. Wu, G., Gao, Z., Dong, A. & Yu, S. Calcium-induced changes in calmodulin structural dynamics and thermodynamics. Int. J. Biol. Macromol. 50, 10111017 (2012).
  52. Porumb, T. Determination of calcium-binding constants by flow dialysis. Anal. Biochem. 220, 227237 (1994).
  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, 38903897 (1981).
  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, 1076810773 (2008).

Download references

Author information

  1. K.W. and A.S. contributed equally to this work

    • Kaihang Wang &
    • Amit Sachdeva

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Kaihang Wang,
    • Amit Sachdeva,
    • Daniel J. Cox,
    • Nabil M. Wilf,
    • Kathrin Lang,
    • Stephen Wallace &
    • Jason W. Chin
  2. Oregon State University, Department of Biochemistry and Biophysics, 2011 ALS, Corvallis, Oregon 97331, USA

    • Ryan A. Mehl

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (18,910 KB)

    Supplementary information

Additional data