An improved cyan fluorescent protein variant useful for FRET

Article metrics


Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Limitations of using ECFP in FRET experiments.
Figure 2: Comparison of Cerulean and ECFP.


  1. 1

    Heim, R. & Tsien, R.Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182 (1996).

  2. 2

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

  3. 3

    Patterson, G.H, Piston, D.W. & Barisas, B.G. Forster distances between green fluorescent protein pairs. Anal. Biochem. 284, 438–440 (2000).

  4. 4

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

  5. 5

    Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

  6. 6

    Ting, A.Y., Kain, K.H., Klemke, R.L. & Tsien, R.Y. Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proc. Natl. Acad. Sci. USA 98, 15003–15008 (2001).

  7. 7

    Zhang, J., Ma, Y., Taylor, S.S. & Tsien, R.Y. Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc. Natl. Acad. Sci. USA 98, 14997–15002 (2001).

  8. 8

    Rizzo, M.A., Magnuson, M.A., Drain, P.F. & Piston, D.W. A functional link between glucokinase binding to insulin granules and conformational alterations in response to glucose and insulin. J. Biol. Chem. 277, 34168–34175 (2002).

  9. 9

    Vanderklish, P.W. et al. Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 97, 2253–2258 (2000).

  10. 10

    Truong, K. et al. FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule. Nat. Struct. Biol. 8, 1069–1073 (2001).

  11. 11

    Swedlow, J.R., Hu, K., Andrews, P.D., Roos, D.S. & Murray, J.M. Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy. Proc. Natl. Acad. Sci. USA 99, 2014–2019 (2002).

  12. 12

    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).

  13. 13

    Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

  14. 14

    Patterson, G., Day, R.N. & Piston, D. Fluorescent protein spectra. J. Cell Sci. 114, 837–838 (2001).

  15. 15

    Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

  16. 16

    Bastiaens, P.I. & Squire, A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9, 48–45 (1999).

  17. 17

    Tramier, M. et al. Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells. Biophys. J. 83, 3570–3577 (2002).

  18. 18

    Hyun Bae, J. et al. Expansion of the genetic code enables design of a novel “gold” class of green fluorescent proteins. J. Mol. Biol. 328, 1071–1081 (2003).

  19. 19

    Cubitt, A.B., Woollenweber, L.A. & Heim, R. Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. Methods Cell Biol. 58, 19–30 (1999).

  20. 20

    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).

Download references


We thank A. Rizzo for helpful suggestions. Funding for this work was provided by US National Institutes of Health grants DK60275 (M.A.R.), and DK53434 and CA86283 (both to D.W.P.), the US National Science Foundation grant BBI-9871063 (D.W.P.), and the US Department of Defense Medical Free-Electron Laser program grant F49620-01-1-0429 (D.W.P.).

Author information

Correspondence to David W Piston.

Ethics declarations

Competing interests

Vanderbilt University has applied for a patent covering the fluorescent proteins described in the article.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Rizzo, M., Springer, G., Granada, B. et al. An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22, 445–449 (2004) doi:10.1038/nbt945

Download citation

Further reading