A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

Abstract

Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of 365 nm and 405 nm, respectively, whereas fluorescence is elicited at 515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

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Figure 1: Properties of Dreiklang.
Figure 2: Molecular basis of Dreiklang photoswitching.
Figure 3: Applications of Dreiklang.
Figure 4: Super-resolution microscopy of living PtK2 cells using Dreiklang.

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References

  1. 1

    Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Lippincott-Schwartz, J., Altan-Bonnet, N. & Patterson, G.H. Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. S7–S14 (2003).

  3. 3

    Lukyanov, K.A., Chudakov, D.M., Lukyanov, S. & Verkhusha, V.V. Innovation: photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6, 885–890 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Dickson, R.M., Cubitt, A.B., Tsien, R.Y. & Moerner, W.E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).

    CAS  Article  Google Scholar 

  5. 5

    Habuchi, S. et al. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc. Natl. Acad. Sci. USA 102, 9511–9516 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Hell, S.W., Jakobs, S. & Kastrup, L. Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl. Phys. A Mater. Sci. Process. 77, 859–860 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Hell, S.W. Toward fluorescence nanoscopy. Nat. Biotechnol. 21, 1347–1355 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S.W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. USA 102, 17565–17569 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Hess, S.T., Girirajan, T.P. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Patterson, G.H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Chudakov, D.M. et al. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22, 1435–1439 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Subach, F.V. et al. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nat. Methods 6, 153–159 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Lukyanov, K.A. et al. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275, 25879–25882 (2000).

    CAS  Article  Google Scholar 

  15. 15

    Cinelli, R.A.G. et al. Green fluorescent proteins as optically controllable elements in bioelectronics. Appl. Phys. Lett. 79, 3353–3355 (2001).

    CAS  Article  Google Scholar 

  16. 16

    Ando, R., Mizuno, H. & Miyawaki, A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Henderson, J.N., Ai, H.W., Campbell, R.E. & Remington, S.J. Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc. Natl. Acad. Sci. USA 104, 6672–6677 (2007).

    CAS  Article  Google Scholar 

  18. 18

    Stiel, A.C. et al. 1.8 Å bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem. J. 402, 35–42 (2007).

    CAS  Article  Google Scholar 

  19. 19

    Andresen, M. et al. Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat. Biotechnol. 26, 1035–1040 (2008).

    CAS  Article  Google Scholar 

  20. 20

    Stiel, A.C. et al. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy. Biophys. J. 95, 2989–2997 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Adam, V. et al. Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. Proc. Natl. Acad. Sci. USA 105, 18343–18348 (2008).

    CAS  Article  Google Scholar 

  22. 22

    Subach, F.V. et al. Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET. Chem. Biol. 17, 745–755 (2010).

    CAS  Article  Google Scholar 

  23. 23

    Sinnecker, D., Voigt, P., Hellwig, N. & Schaefer, M. Reversible photobleaching of enhanced green fluorescent proteins. Biochemistry 44, 7085–7094 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Shaner, N.C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008).

    CAS  Article  Google Scholar 

  25. 25

    Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Article  Google Scholar 

  26. 26

    Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein – Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    CAS  Article  Google Scholar 

  27. 27

    Siegbahn, P.E.M., Wirstam, M. & Zimmer, M. Theoretical study of the mechanism of peptide ring formation in green fluorescent protein. Int. J. Quantum Chem. 81, 169–186 (2001).

    CAS  Article  Google Scholar 

  28. 28

    Rosenow, M.A., Huffman, H.A., Phail, M.E. & Wachter, R.M. The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation. Biochemistry 43, 4464–4472 (2004).

    CAS  Article  Google Scholar 

  29. 29

    Barondeau, D.P., Kassmann, C.J., Tainer, J.A. & Getzoff, E.D. Understanding GFP chromophore biosynthesis: controlling backbone cyclization and modifying post-translational chemistry. Biochemistry 44, 1960–1970 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Irie, M., Fukaminato, T., Sasaki, T., Tamai, N. & Kawai, T. Organic chemistry: a digital fluorescent molecular photoswitch. Nature 420, 759–760 (2002).

    CAS  Article  Google Scholar 

  31. 31

    Sauer, M. Reversible molecular photoswitches: a key technology for nanoscience and fluorescence imaging. Proc. Natl. Acad. Sci. USA 102, 9433–9434 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2, 444–456 (2001).

    CAS  Article  Google Scholar 

  33. 33

    Phair, R.D. & Misteli, T. Kinetic modelling approaches to in vivo imaging. Nat. Rev. Mol. Cell Biol. 2, 898–907 (2001).

    CAS  Article  Google Scholar 

  34. 34

    Sprague, B.L. & McNally, J.G. FRAP analysis of binding: proper and fitting. Trends Cell Biol. 15, 84–91 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Chudakov, D.M., Chepurnykh, T.V., Belousov, V.V., Lukyanov, S. & Lukyanov, K.A. Fast and precise protein tracking using repeated reversible photoactivation. Traffic 7, 1304–1310 (2006).

    CAS  Article  Google Scholar 

  36. 36

    Hell, S.W. Microscopy and its focal switch. Nat. Methods 6, 24–32 (2009).

    CAS  Article  Google Scholar 

  37. 37

    Rust, M., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    CAS  Article  Google Scholar 

  38. 38

    Lamesch, P. et al. hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes. Genomics 89, 307–315 (2007).

    CAS  Article  Google Scholar 

  39. 39

    Testa, I. et al. Nanoscale separation of molecular species based on their rotational mobility. Opt. Express 16, 21093–21104 (2008).

    CAS  Article  Google Scholar 

  40. 40

    Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R. & Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).

    CAS  Article  Google Scholar 

  41. 41

    Bossi, M. et al. Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett. 8, 2463–2468 (2008).

    CAS  Article  Google Scholar 

  42. 42

    Richter, F.M., Sander, B., Golas, M.M., Stark, H. & Urlaub, H. Merging molecular electron microscopy and mass spectrometry by carbon film-assisted endoproteinase digestion. Mol. Cell. Proteomics 9, 1729–1741 (2010).

    CAS  Article  Google Scholar 

  43. 43

    Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993).

    CAS  Article  Google Scholar 

  44. 44

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007).

    CAS  Article  Google Scholar 

  45. 45

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  46. 46

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  47. 47

    Schuttelkopf, A.W. & van Aalten, D.M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  Google Scholar 

  48. 48

    Moriarty, N.W., Grosse-Kunstleve, R.W. & Adams, P.D. electronic ligand builder and optimization workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr. 65, 1074–1080 (2009).

    CAS  Article  Google Scholar 

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Acknowledgements

We acknowledge access to beamline BL14.2 of the BESSY II storage ring (Berlin) through the Joint Berlin MX-Laboratory sponsored by the Helmholtz Zentrum Berlin für Materialien und Energie, the Freie Universität Berlin, the Humboldt-Universität zu Berlin, the Max-Delbrück Centrum and the Leibniz-Institut für Molekulare Pharmakologie. We thank V. Belov for insightful discussions and F. Lavoie-Cardinal for help with the set-up for targeted switching. We acknowledge A. Schönle for providing the software ImSpector. We also thank T. Gilat, S. Löbermann, R. Pick and E. Rothermel for excellent technical assistance, H.-H. Hsiao for help in ESI-MS and H. Schill and J. Jethwa for carefully reading the manuscript. We acknowledge R.Y. Tsien for sharing the plasmid pRSET-Citrine. This work was supported by the Deutsche Forschungsgemeinschaft through the Gottfried Wilhelm Leibniz Prize (to S.W.H.) and through the DFG-Research Center for Molecular Physiology of the Brain (to S.J.).

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G.W., M.A. and I.T. contributed equally to this work. C.E., S.W.H. and S.J. conceived the project. T.B., A.C.S., G.W., M.A., I.T., T.G., M.L. and U.P. performed all experiments. I.T. recorded the super-resolution images. Data analysis was done by T.B., A.C.S., G.W., M.A., I.T., T.G., M.L., H.U., C.E., M.C.W., S.W.H. and S.J. The manuscript was written by S.W.H. and S.J. All authors discussed the results and commented on the manuscript.

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Correspondence to Stefan W Hell or Stefan Jakobs.

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A patent application concerning the protein Dreiklang has been filed.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1,2 and Supplementary and Figures 1–17 (PDF 1847 kb)

Supplementary Movie 1

Animated sequence of 33 individual images as shown in Figure 3a. (MPG 232 kb)

Supplementary Movie 2

Animated sequence of 100 consecutive switching cycles of vimentin-Dreiklang in PtK2 cells, as shown in Supplementary Figure 13. (AVI 1396 kb)

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Brakemann, T., Stiel, A., Weber, G. et al. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat Biotechnol 29, 942–947 (2011). https://doi.org/10.1038/nbt.1952

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