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.

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Abstract

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions1,2,3,4,5. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: In vitro evolution of a chromoprotein from the stony coral Montipora sp. into a fluorescent protein with a large Stokes shift.
Figure 2: Single laser wavelength (458 nm) excitation FCCS using mKeima and CFP to monitor proteolysis by caspase-3.
Figure 3: Single laser wavelength (458 nm) excitation FCCS using mKeima and CFP to monitor the Ca2+-dependent association between CaM and CaMKI.
Figure 4: Simultaneous six-color imaging of subcellular structures in a Vero cell using a single laser line (458 nm).

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 1

    Kettling, U., Koltermann, A., Schwille, P. & Eigen, M. Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc. Natl. Acad. Sci. USA 95, 1416–1420 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Weidemann, T., Wachsmuth, M., Tewes, M., Rippe, K. & Langowski, J. Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy. Single Mol. 3, 49–61 (2002).

    CAS  Article  Google Scholar 

  3. 3

    Kim, S.A. & Schwille, P. Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience. Curr. Opin. Neurobiol. 13, 583–590 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Saito, K., Wada, I., Tamura, M. & Kinjo, M. Direct detection of caspase-3 activation in single live cells by cross-correlation analysis. Biochem. Biophys. Res. Commun. 324, 849–854 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Kohl, T., Haustein, E. & Schwille, P. Determining protease activity in vivo by fluorescence cross-correlation analysis. Biophys. J. 89, 2770–2782 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Hwang, L.C. & Wohland, T. Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: Resolution for binding studies. J. Chem. Phys. 122, 114708 (1–11) (2005).

    Article  Google Scholar 

  7. 7

    Martin, B.R., Giepmans, B.N., Adams, S.R. & Tsien, R.Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Helmchen, F. & Denk, W. New developments in multiphoton microscopy. Curr. Opin. Neurobiol. 12, 593–601 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Heinze, K.G., Koltermann, A. & Schwille, P. Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).

    CAS  Article  Google Scholar 

  10. 10

    Kohl, T., Heinze, K.G., Kuhlemann, R., Koltermann, A. & Schwille, P. A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl. Acad. Sci. USA 99, 12161–12166 (2002).

    CAS  Article  Google Scholar 

  11. 11

    Heinze, K.G., Rarbach, M., Jahnz, M. & Schwille, P. Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics. Biophys. J. 83, 1671–1681 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Kim, S.A., Heinze, K.G., Waxham, M.N. & Schwille, P. Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc. Natl. Acad. Sci. USA 101, 105–110 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Patterson, G.H. & Piston, D.W. Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Chen, T.S., Zeng, S.Q., Luo, Q.M., Zhang, Z.H. & Zhou, W. High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy. Biophys. Biochem. Res. Commun. 291, 1272–1275 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Matz, M.V., Lukyanov, K.A. & Lukyanov, S.A. Family of the green fluorescent protein: journey to the end of the rainbow. Bioessays 24, 953–959 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Labas, Y.A. et al. Diversity and evolution of the green fluorescent protein family. Proc. Natl. Acad. Sci. USA 99, 4256–4261 (2002).

    CAS  Article  Google Scholar 

  17. 17

    Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N. & Miyawaki, A. Semi-rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6, 233–238 (2005).

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

    Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).

    CAS  Article  Google Scholar 

  20. 20

    Crivici, A. & Ikura, M. Molecular and structural basis of target recognition by calmodulin. Annu. Rev. Biophys. Biomol. Struct. 24, 85–116 (1995).

    CAS  Article  Google Scholar 

  21. 21

    Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554–10559 (2004).

    CAS  Article  Google Scholar 

  22. 22

    Karasawa, S., Araki, T., Nagai, T., Mizuno, H. & Miyawaki, A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem. J. 381, 307–312 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Karasawa, S., Araki, T., Yamamoto-Hino, M. & Miyawaki, A. A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling. J. Biol. Chem. 278, 34167–34171 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Sawano, A. & Miyawaki, A. Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 28, E78 (2000).

    CAS  Article  Google Scholar 

  25. 25

    Arscott, P.L. et al. Fas (CD95) expression is up-regulated on papillary thyroid carcinoma. J. Clin. Endocrinol. Metab. 84, 4246–4252 (1999).

    CAS  PubMed  Google Scholar 

  26. 26

    Llopis, J., McCaffery, J.M., Miyawaki, A., Farquhar, M.G. & Tsien, R.Y. Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc. Natl. Acad. Sci. USA 95, 6803–6808 (1998).

    CAS  Article  Google Scholar 

  27. 27

    Sato, S. et al. Aberrant tau phosphorylation by glycogen synthase kinase-3b and JNK3 induces oligomeric tau fibrils in COS-7 cells. J. Biol. Chem. 277, 2060–2065 (2002).

    Google Scholar 

  28. 28

    Sawano, A., Hama, H., Saito, N. & Miyawaki, A. Multicolor imaging of Ca2+ and protein kinase C signals using novel epifluorescence microscopy. Biophys. J. 82, 1076–1085 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Mochizuki, N. et al. Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature 411, 1065–1068 (2001).

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors would like to thank K. Iwao and S. Hosaka at the Akajima Marine Science Laboratory for acquiring the stony coral animals, Y. Isogai for assistance with analytical centrifugation, F. Ishidate, K. Weisshart, B. Zimmerman, Y. Hasegawa for assistance with FCCS measurements and spectral imaging, and K. Ishihara, H. Watanabe, T. Fukano, and M. Hirano for assistance with multi-color imaging and fluorescence lifetime measurements. This work was partly supported by grants from Japan MEXT Grant-in-Aid for Scientific Research on priority areas, NEDO (the New Energy and Industrial Technology Development Organization), HFSP (the Human Frontier Science Program), and RIKEN Strategic Research Program.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Atsushi Miyawaki.

Ethics declarations

Competing interests

S.K. and T.A. get salaries from Amalgaam and Medical Biological Laboratories, which will sell the products presented in this paper.

Supplementary information

Supplementary Fig. 1

An amino-acid sequence alignment of DsRed with #20, #20-9115, tKeima, dKeima, mKeima, and dKeima570. (PDF 194 kb)

Supplementary Fig. 2

Equilibrium radial absorbance profiles obtained by analytical ultracentrifugation for Keima variants. (PDF 288 kb)

Supplementary Fig. 3

Quantitative performance of FCCS using an mKeima-CFP pair. (PDF 262 kb)

Supplementary Fig. 4

Detection of the Ca2+-dependent association between CaM and CaMKI with the SL-FCCS technique using mKeima and CFP. (PDF 219 kb)

Supplementary Fig. 5

Single laser wavelength (458 nm) excitation FCCS to monitor the Ca2+-dependent association between CaM and M13 in a living HeLa cell, which was expressing mCFP-CaM and M13-mKeima. (PDF 251 kb)

Supplementary Fig. 6

Simultaneous imaging of [Ca2+]c and mitochondrial morphology. (PDF 937 kb)

Supplementary Fig. 7

Excitation (broken lines) and emission (solid lines) spectra of dKeima and dKeima570. (PDF 215 kb)

Supplementary Table 1

Spectral characteristics of CFP, Keima, dKeima, mKeima, and dKeima570. (PDF 85 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kogure, T., Karasawa, S., Araki, T. et al. A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy. Nat Biotechnol 24, 577–581 (2006). https://doi.org/10.1038/nbt1207

Download citation

Further reading

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