Bright cyan fluorescent protein variants identified by fluorescence lifetime screening

Abstract

Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.

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Figure 1: Fluorescence lifetime screening and spectral properties of cyan fluorescent protein variants.
Figure 2: Three-component lifetime unmixing of cyan fluorescent protein variants from a single (frequency) FLIM experiment.

References

  1. 1

    Heim, R. & Tsien, R.Y. Curr. Biol. 6, 178–182 (1996).

    CAS  Article  Google Scholar 

  2. 2

    Chudakov, D.M., Lukyanov, S. & Lukyanov, K.A. Trends Biotechnol. 23, 605–613 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Miyawaki, A. Dev. Cell 4, 295–305 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Kremers, G.J., Goedhart, J., van Munster, E.B. & Gadella, T.W. Jr. Biochemistry 45, 6570–6580 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. Nat. Biotechnol. 22, 445–449 (2004).

    CAS  Article  Google Scholar 

  6. 6

    van Munster, E.B. & Gadella, T.W. Adv. Biochem. Eng. Biotechnol. 95, 143–175 (2005).

    PubMed  Google Scholar 

  7. 7

    Merzlyak, E.M. et al. Nat. Methods 4, 555–557 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Mena, M.A., Treynor, T.P., Mayo, S.L. & Daugherty, P.S. Nat. Biotechnol. 24, 1569–1571 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Nguyen, A.W. & Daugherty, P.S. Nat. Biotechnol. 23, 355–360 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Chen, Y., Muller, J.D., So, P.T. & Gratton, E. Biophys. J. 77, 553–567 (1999).

    CAS  Article  Google Scholar 

  11. 11

    Szymczak, A.L. et al. Nat. Biotechnol. 22, 589–594 (2004).

    CAS  Article  Google Scholar 

  12. 12

    Digman, M.A., Caiolfa, V.R., Zamai, M. & Gratton, E. Biophys. J. 94, L14–L16 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Pepperkok, R., Squire, A., Geley, S. & Bastiaens, P.I. Curr. Biol. 9, 269–272 (1999).

    CAS  Article  Google Scholar 

  14. 14

    Verveer, P.J., Wouters, F.S., Reynolds, A.R. & Bastiaens, P.I. Science 290, 1567–1570 (2000).

    CAS  Article  Google Scholar 

  15. 15

    Yasuda, R. et al. Nat. Neurosci. 9, 283–291 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Riedl, J. et al. Nat. Methods 5, 605–607 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Vermeer, J.E., Van Munster, E.B., Vischer, N.O. & Gadella, T.W. Jr. J. Microsc. 214, 190–200 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Bacia, K. & Schwille, P. Methods 29, 74–85 (2003).

    CAS  Article  Google Scholar 

  19. 19

    Perroud, T.D., Huang, B. & Zare, R.N. ChemPhysChem 6, 905–912 (2005).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank I. Elzenaar and M. Adjobo-Hermans for pilot experiments on the 2A co-expression assay and the members of our laboratory for encouraging discussions, J.E.M. Vermeer (University of Amsterdam) for providing plasmid encoding peroxi-YFP with a C-terminal SKL peroxisomal targeting sequence and E.L. Snapp (Albert Einstein College of Medicine) for providing plasmid encoding ER-tdTomato. Part of this work is supported by the EU integrated project on 'Molecular Imaging' (LSHG-CT-2003-503259).

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J.G. and T.W.J.G. designed research; J.G., L.v.W., M.A.H., K.J. and T.W.J.G. conducted experiments; J.G., L.v.W., M.A.H., N.O.E.V. and T.W.J.G. analyzed data; and J.G. and T.W.J.G. wrote the paper.

Corresponding author

Correspondence to Theodorus W J Gadella Jr.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6, Supplementary Table 1, Supplementary Note and Supplementary Data (PDF 4861 kb)

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Goedhart, J., van Weeren, L., Hink, M. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7, 137–139 (2010). https://doi.org/10.1038/nmeth.1415

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