Journal of Biological Physics

, Volume 31, Issue 3–4, pp 249–259 | Cite as

Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein

  • Sergey Ivanchenko
  • Carlheinz Röcker
  • Franz Oswald
  • Jörg Wiedenmann
  • G. Ulrich NienhausEmail author


EosFP is a novel fluorescent protein from the stony coral Lobophyllia hemprichii. Its gene was cloned in Escherichia coli to express the tetrameric wild-type protein. The protein emits strong green fluorescence (516 nm) that shifts toward red (581 nm) upon near-ultraviolet irradiation at ∼390 nm due to a photo-induced modification that involves a break in the peptide backbone next to the chromophore. Using site-directed mutagenesis, dimeric (d1EosFP, d2EosFP) and monomeric (mEosFP) variants were produced with essentially unaltered spectroscopic properties. Here we present a spectroscopic characterization of EosFP and its variants, including room- and low-temperature spectra, fluorescence lifetime determinations, two-photon excitation and two-photon photoconversion. Furthermore, by transfection of a human cancer (HeLa) cell with a fusion construct of a mitochondrial targeting sequence and d2EosFP, we demonstrate how localized photoconversion of EosFP can be employed for resolving intracellular processes.

Key words

fluorescent protein fusion protein two-photon excitation fluorescence spectroscopy photoconversion 


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  1. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. and Cormier, M.J.: Primary Structure of the Aequorea Victoria Green-Fluorescent Protein, Gene 111 (1992), 229–233.CrossRefGoogle Scholar
  2. Ormö, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y. and Remington, S.J.: Crystal Structure of the Aequorea Victoria Green Fluorescent Protein, Science 273 (1996), 1392–1395.ADSGoogle Scholar
  3. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W. and Prasher, D.C.: Green Fluorescent Protein as a Marker for Gene Expression, Science 263 (1994), 802–805.ADSGoogle Scholar
  4. Tsien, R.Y.: The Green Fluorescent Protein, Annu. Rev. Biochem. 67 (1998), 509–544.CrossRefGoogle Scholar
  5. Zhang, J., Campbell, R.E., Ting, A.Y. and Tsien, R.Y.: Creating New Fluorescent Probes for Cell Biology, Nat. Rev. Mol. Cell. Biol. 3 (2002), 906–918.CrossRefGoogle Scholar
  6. Wiedenmann, J.: In Offenlegungsschrift DE 197 18 640 A1, Deutsches Patent-und Markenamt, Munich, Germany, 1997, pp 1–18.Google Scholar
  7. Matz, M.V., Fradkov, A.F., Labas, Y.A., Savitsky, A.P., Zaraisky, A.G., Markelov, M.L. and Lukyanov, S.A.: Fluorescent Proteins from Nonbioluminescent Anthozoa Species, Nat. Biotechnol. 17 (1999), 969–973.CrossRefGoogle Scholar
  8. Fradkov, A.F., Chen, Y., Ding, L., Barsova, E.V., Matz, M.V. and Lukyanov, S.A.: Novel Fluorescent Protein from Discosoma Coral and its Mutants Possesses a Unique Far-Red Fluorescence, FEBS Lett. 479 (2000), 127–130.CrossRefGoogle Scholar
  9. Wiedenmann, J., Elke, C., Spindler, K.D. and Funke, W.: Cracks in the beta-can: Fluorescent Proteins from Anemonia Sulcata (Anthozoa, Actinaria), Proc. Natl. Acad. Sci. U.S.A. 97 (2000), 14091–14096.CrossRefADSGoogle Scholar
  10. Wiedenmann, J., Ivanchenko, S., Oswald, F. and Nienhaus, G.U.: Identification of GFP-like Proteins in Nonbioluminescent, Azooxanthellate Anthozoa Opens New Perspectives for Bioprospecting, Mar. Biotechnol. (N.Y.) 6 (2004), 270–277.Google Scholar
  11. Wiedenmann, J., Schenk, A., Röcker, C., Girod, A., Spindler, K.D. and Nienhaus, G.U.: A Far-Red Fluorescent Protein with Fast Maturation and Reduced Oligomerization Tendency from Entacmaea Quadricolor (Anthozoa, Actinaria), Proc. Natl. Acad. Sci. U.S.A. 99 (2002), 11646–11651.CrossRefADSGoogle Scholar
  12. Schenk, A., Ivanchenko, S., Röcker, C., Wiedenmann, J. and Nienhaus, G.U.: Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy, Biophys. J. 86 (2004), 384–394.Google Scholar
  13. Baird, G.S., Zacharias, D.A. and Tsien, R.Y.: Biochemistry, Mutagenesis, and Oligomerization of DsRed, a Red Fluorescent Protein from Coral, Proc. Natl. Acad. Sci. U.S.A. 97 (2000), 11984–11989.ADSGoogle Scholar
  14. Wall, M.A., Socolich, M. and Ranganathan, R.: The Structural Basis for Red Fluorescence in the Tetrameric GFP Homolog DsRed, Nat. Struct. Biol. 7 (2000), 1133–1138.Google Scholar
  15. Yarbrough, D., Wachter, R.M., Kallio, K., Matz, M.V. and Remington, S.J.: Refined Crystal Structure of DsRed, a Red Fluorescent Protein from Coral, at 2.0-A Resolution, Proc. Natl. Acad. Sci. U.S.A. 98 (2001), 462–467.CrossRefADSGoogle Scholar
  16. Nienhaus, K., Vallone, B., Renzi, F., Wiedenmann, J. and Nienhaus, G.U.: Crystallization and Preliminary X-ray Diffraction Analysis of the Red Fluorescent Protein eqFP611, Acta Crystallogr. D Biol. Crystallogr. 59 (2003), 1253–1255.CrossRefGoogle Scholar
  17. Shagin, D.A., Barsova, E.V., Yanushevich, Y.G., Fradkov, A.F., Lukyanov, K.A., Labas, Y.A., Semenova, T.N., Ugalde, J.A., Meyers, A., Nunez, J.M., Widder, E.A., Lukyanov, S.A. and Matz, M.V.: GFP-like Proteins as Ubiquitous Metazoan Superfamily: Evolution of Functional Features and Structural Complexity, Mol. Biol. Evol. 21 (2004), 841–850.Google Scholar
  18. Lippincott-Schwartz, J. and Patterson, G.H.: Development and Use of Fluorescent Protein Markers in Living Cells, Science 300 (2003), 87–91.CrossRefADSGoogle Scholar
  19. Patterson, G.H. and Lippincott-Schwartz, J.: A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells, Science 297 (2002), 1873–1877.CrossRefADSGoogle Scholar
  20. Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S. and Lukyanov, K.A.: Kindling Fluorescent Proteins for Precise In Vivo Photolabeling, Nat. Biotechnol. 21 (2003), 191–194.CrossRefGoogle Scholar
  21. Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H. and Miyawaki, A.: An Optical Marker Based on the UV-Induced Green-to-Red Photoconversion of a Fluorescent Protein, Proc. Natl. Acad. Sci. U.S.A. 99 (2002), 12651–12656.CrossRefADSGoogle Scholar
  22. Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Röcker, C., Salih, A., Spindler, K.D. and Nienhaus, G.U.: EosFP, a Fluorescent Marker Protein with UV-Inducible Green-to-Red Fluorescence Conversion, Proc. Natl. Acad. Sci. U.S.A. 101 (2004), 15905–15910.CrossRefADSGoogle Scholar
  23. Rizzuto, R., Brini, M., Pizzo, P., Murgia, M. and Pozzan, T.: Chimeric Green Fluorescent Protein as a Tool for Visualizing Subcellular Organelles in Living Cells, Curr. Biol. 5 (1995), 635–642.CrossRefGoogle Scholar
  24. Mizuno, H., Mal, T.K., Tong, K.I., Ando, R., Furuta, T., Ikura, M. and Miyawaki, A.: Photo-Induced Peptide Cleavage in the Green-to-Red Conversion of a Fluorescent Protein, Mol. Cell 12 (2003), 1051–1058.CrossRefGoogle Scholar
  25. Albota, M.A., Xu, C. and Webb, W.W.: Two-Photon Fluorescence Excitation Cross Sections of Biomolecular Probes from 690 to 960 nm, Appl. Opt. 37 (1998), 7352–7356.ADSCrossRefGoogle Scholar
  26. Xu, C., Zipfel, W., Shear, J.B., Williams, R.M. and Webb, W.W.: Multiphoton Fluorescence Excitation: New Spectral Windows for Biological Nonlinear Microscopy, Proc. Natl. Acad. Sci. U.S.A. 93 (1996), 10763–10768.ADSGoogle Scholar
  27. Schwille, P., Haupts, U., Maiti, S. and Webb, W.W.: Molecular Dynamics in Living Cells Observed by Fluorescence Correlation Spectroscopy with One- and Two-Photon Excitation, Biophys. J. 77 (1999), 2251–2265.CrossRefGoogle Scholar
  28. Collins, T.J., Berridge, M.J., Lipp, P. and Bootman, M.D.: Mitochondria are Morphologically and Functionally Heterogeneous Within Cells, EMBO J. 21 (2002), 1616–1627.CrossRefGoogle Scholar
  29. Arimura, S., Yamamoto, J., Aida, G.P., Nakazono, M. and Tsutsumi, N.: Frequent Fusion and Fission of Plant Mitochondria with Unequal Nucleoid Distribution, Proc. Natl. Acad. Sci. U.S.A. 101 (2004), 7805–7808.CrossRefADSGoogle Scholar
  30. Jakobs, S., Schauss, A.C. and Hell, S.W.: Photoconversion of Matrix Targeted GFP Enables Analysis of Continuity and Intermixing of the Mitochondrial Lumen, FEBS Lett. 554 (2003), 194–200.CrossRefGoogle Scholar
  31. Wiedenmann, J., Vallone, B., Renzi, F., Nienhaus, K., Ivanchenko, S., Röcker, C. and Nienhaus, G.U.: The Red Fluorescent Protein eqFP611 and its Genetically Engineered Dimeric Variants, J. Biomed. Opt. 10 (2005), 14003.CrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Sergey Ivanchenko
    • 1
  • Carlheinz Röcker
    • 1
  • Franz Oswald
    • 2
  • Jörg Wiedenmann
    • 3
  • G. Ulrich Nienhaus
    • 1
    • 4
    Email author
  1. 1.Department of BiophysicsUniversity of UlmUlmGermany
  2. 2.Department of Internal Medicine IUniversity of UlmUlmGermany
  3. 3.Department of General Zoology and EndocrinologyUniversity of UlmUlmGermany
  4. 4.Department of PhysicsUniversity of Illinois at Urbana-ChampaignUrbana

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