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.

  • Article
  • Published:

Small-molecule control of protein function through Staudinger reduction

Nature Chemistry volume 8, pages 1027–1034 (2016)Cite this article

Subjects

Abstract

Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).

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: Staudinger reduction-based protein activation through genetic encoding of OABK in living cells.
Figure 2: Small-molecule-induced activation of EGFP fluorescence.
Figure 3: Phosphine screening for Staudinger reduction-mediated deprotection and activation of FLuc in cells and cell lysates.
Figure 4: Small-molecule-triggered protein translocation.
Figure 5: Small-molecule-triggered DNA recombination.
Figure 6: Small-molecule-triggered CRISPR/Cas9 gene editing.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Bishop, A. C. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 (2000).

    CAS  PubMed  Google Scholar 

  2. Buckley, D. L. & Crews, C. M. Small-molecule control of intracellular protein levels through modulation of the ubiquitin proteasome system. Angew. Chem. Int. Ed. 53, 2312–2330 (2014).

    CAS  Google Scholar 

  3. Winter, G. E. et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Warner, J. B., Muthusamy, A. K. & Petersson, E. J. Specific modulation of protein activity by using a bioorthogonal reaction. Chembiochem 15, 2508–2514 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Buskirk, A. R. & Liu, D. R. Creating small-molecule-dependent switches to modulate biological functions. Chem. Biol. 12, 151–161 (2005).

    CAS  PubMed  Google Scholar 

  6. Zorn, J. A. & Wells, J. A. Turning enzymes ON with small molecules. Nature Chem. Biol. 6, 179–188 (2010).

    CAS  Google Scholar 

  7. Putyrski, M. & Schultz, C. Protein translocation as a tool: the current rapamycin story. FEBS Lett. 586, 2097–2105 (2012).

    CAS  PubMed  Google Scholar 

  8. Fegan, A., White, B., Carlson, J. C. & Wagner, C. R. Chemically controlled protein assembly: techniques and applications. Chem. Rev. 110, 3315–3336 (2010).

    CAS  PubMed  Google Scholar 

  9. Rakhit, R., Navarro, R. & Wandless, T. J. Chemical biology strategies for posttranslational control of protein function. Chem. Biol. 21, 1238–1252 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Ho, S. N., Biggar, S. R., Spencer, D. M., Schreiber, S. L. & Crabtree, G. R. Dimeric ligands define a role for transcriptional activation domains in reinitiation. Nature 382, 822–826 (1996).

    CAS  PubMed  Google Scholar 

  11. Gray, D. C., Mahrus, S. & Wells, J. A. Activation of specific apoptotic caspases with an engineered small-molecule-activated protease. Cell 142, 637–646 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V. & Hahn, K. M. Engineered allosteric activation of kinases in living cells. Nature Biotechnol. 28, 743–747 (2010).

    CAS  Google Scholar 

  13. Jullien, N., Sampieri, F., Enjalbert, A. & Herman, J. P. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 31, e131 (2003).

    PubMed  PubMed Central  Google Scholar 

  14. Bonger, K. M., Chen, L. C., Liu, C. W. & Wandless, T. J. Small-molecule displacement of a cryptic degron causes conditional protein degradation. Nature Chem. Biol. 7, 531–537 (2011).

    CAS  Google Scholar 

  15. Chu, P. H. et al. Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms. Proc. Natl Acad. Sci. USA 111, 12420–12425 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Miyamoto, T. et al. Rapid and orthogonal logic gating with a gibberellin-induced dimerization system. Nature Chem. Biol. 8, 465–470 (2012).

    CAS  Google Scholar 

  17. Gautier, A. et al. How to control proteins with light in living systems. Nature Chem. Biol. 10, 533–541 (2014).

    CAS  Google Scholar 

  18. Weitzman, M. & Hahn, K. M. Optogenetic approaches to cell migration and beyond. Curr. Opin. Cell Biol. 30, 112–120 (2014).

    CAS  PubMed  Google Scholar 

  19. Chen, X. et al. Synthetic dual-input mammalian genetic circuits enable tunable and stringent transcription control by chemical and light. Nucleic Acids Res. 44, 2677–2690 (2016).

    PubMed  Google Scholar 

  20. Jeong, J. W. et al. Wireless optofluidic systems for programmable in vivo pharmacology and optogenetics. Cell 162, 662–674 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Li, J., Jia, S. & Chen, P. R. Diels–Alder reaction-triggered bioorthogonal protein decaging in living cells. Nature Chem. Biol. 10, 1003–1005 (2014).

    CAS  Google Scholar 

  22. Hemphill, J., Chou, C. J., Chin, J. W. & Deiters, A. Genetically encoded light-activated transcription for spatiotemporal control of gene expression and gene silencing in mammalian cells. J. Am. Chem. Soc. 135, 13433–13439 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, H., Hah, J. M. & Lawrence, D. S. Light-mediated liberation of enzymatic activity: ‘small molecule’ caged protein equivalents. J. Am. Chem. Soc. 130, 10474–10475 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Li, J. et al. Palladium-triggered deprotection chemistry for protein activation in living cells. Nature Chem. 6, 352–361 (2014).

    CAS  Google Scholar 

  25. Vila-Perello, M., Hori, Y., Ribo, M. & Muir, T. W. Activation of protein splicing by protease- or light-triggered O to N acyl migration. Angew. Chem. Int. Ed. 47, 7764–7767 (2008).

    CAS  Google Scholar 

  26. Sainlos, M., Iskenderian-Epps, W. S., Olivier, N. B., Choquet, D. & Imperiali, B. Caged mono- and divalent ligands for light-assisted disruption of PDZ domain-mediated interactions. J. Am. Chem. Soc. 135, 4580–4583 (2013).

    CAS  PubMed  Google Scholar 

  27. Kohn, M. & Breinbauer, R. The Staudinger ligation—a gift to chemical biology. Angew. Chem. Int. Ed. 43, 3106–3116 (2004).

    Google Scholar 

  28. Van Berkel, S. S., Van Eldijk, M. B. & Van Hest, J. C. Staudinger ligation as a method for bioconjugation. Angew. Chem. Int. Ed. 50, 8806–8827 (2011).

    CAS  Google Scholar 

  29. Sletten, E. M. & Bertozzi, C. R. From mechanism to mouse: a tale of two bioorthogonal reactions. Acc. Chem. Res. 44, 666–676 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Alouane, A., Labruere, R., Le Saux, T., Schmidt, F. & Jullien, L. Self-immolative spacers: kinetic aspects, structure–property relationships, and applications. Angew. Chem. Int. Ed. 54, 7492–7509 (2015).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  32. Takimoto, J. K., Dellas, N., Noel, J. P. & Wang, L. Stereochemical basis for engineered pyrrolysyl-tRNA synthetase and the efficient in vivo incorporation of structurally divergent non-native amino acids. ACS Chem. Biol. 6, 733–743 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Google Scholar 

  35. Wang, Y. S., Fang, X. Q., Wallace, A. L., Wu, B. & Liu, W. S. R. A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum. J. Am. Chem. Soc. 134, 2950–2953 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  37. Luo, J. et al. Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation. J. Am. Chem. Soc. 136, 15551–15558 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Iizuka, R., Yamagishi-Shirasaki, M. & Funatsu, T. Kinetic study of de novo chromophore maturation of fluorescent proteins. Anal. Biochem. 414, 173–178 (2011).

    CAS  PubMed  Google Scholar 

  39. Kawai, K. et al. A reductant-resistant and metal-free fluorescent probe for nitroxyl applicable to living cells. J. Am. Chem. Soc. 135, 12690–12696 (2013).

    CAS  PubMed  Google Scholar 

  40. Saneyoshi, H. et al. Triphenylphosphinecarboxamide: an effective reagent for the reduction of azides and its application to nucleic acid detection. Org. Lett. 16, 30–33 (2014).

    CAS  PubMed  Google Scholar 

  41. Cline, D. J. et al. New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability. Biochemistry 43, 15195–15203 (2004).

    CAS  PubMed  Google Scholar 

  42. Baker, A. S. & Deiters, A. Optical control of protein function through unnatural amino acid mutagenesis and other optogenetic approaches. ACS Chem. Biol. 9, 1398–1407 (2014).

    CAS  PubMed  Google Scholar 

  43. Brieke, C., Rohrbach, F., Gottschalk, A., Mayer, G. & Heckel, A. Light-controlled tools. Angew. Chem. Int. Ed. 51, 8446–8476 (2012).

    CAS  Google Scholar 

  44. Engelke, H., Chou, C., Uprety, R., Jess, P. & Deiters, A. Control of protein function through optochemical translocation. ACS Synth. Biol. 3, 731–736 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Ho, P. S. & Eichman, B. F. The crystal structures of DNA Holliday junctions. Curr. Opin. Struct. Biol. 11, 302–308 (2001).

    CAS  PubMed  Google Scholar 

  46. Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).

    CAS  PubMed  Google Scholar 

  47. Gibb, B. et al. Requirements for catalysis in the Cre recombinase active site. Nucleic Acids Res. 38, 5817–5832 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Yang, Y. S. & Hughes, T. E. Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells. Biotechniques 31, 1036–1041 (2001).

    CAS  PubMed  Google Scholar 

  49. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A. & Liu, D. R. Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nature Chem. Biol. 11, 316–318 (2015).

    CAS  Google Scholar 

  51. Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nature Biotechnol. 33, 139–142 (2015).

    CAS  Google Scholar 

  52. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Hemphill, J., Borchardt, E. K., Brown, K., Asokan, A. & Deiters, A. Optical control of CRISPR/Cas9 gene editing. J. Am. Chem. Soc. 137, 5642–5645 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported in part by the National Institutes of Health (1R01GM112728), the National Science Foundation (MCB-1330746) and the Charles E. Kaufman Foundation of The Pittsburgh Foundation. K.M. is grateful for a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad. We thank the Chin lab (Medical Research Council) for plasmids encoding the PylRS and PylT genes, the Asokan lab (University of North Carolina) for the pgRNA and pIRG plasmids and the Hughes lab (Montana State University) for the Cre Stoplight plasmid.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Pittsburgh, Pittsburgh, 15260, Pennsylvania, USA

    Ji Luo, Qingyang Liu, Kunihiko Morihiro & Alexander Deiters

Authors
  1. Ji Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qingyang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kunihiko Morihiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander Deiters
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.D. and J.L. conceived and designed the experiments. J.L, Q.L. and K.M. performed the experiments and analysed the data. A.D. and J.L. co-wrote the paper.

Corresponding author

Correspondence to Alexander Deiters.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 2316 kb)

Supplementary information

Supplementary Movie 1 (MP4 2417 kb)

Supplementary information

Supplementary Movie 2 (MP4 999 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Luo, J., Liu, Q., Morihiro, K. et al. Small-molecule control of protein function through Staudinger reduction. Nature Chem 8, 1027–1034 (2016). https://doi.org/10.1038/nchem.2573

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchem.2573

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