Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein
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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 wordsfluorescent protein fusion protein two-photon excitation fluorescence spectroscopy photoconversion
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- Wiedenmann, J.: In Offenlegungsschrift DE 197 18 640 A1, Deutsches Patent-und Markenamt, Munich, Germany, 1997, pp 1–18.Google Scholar
- 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
- 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
- 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
- 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
- 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