Targeted green-red photoconversion of EosFP, a fluorescent marker protein

Ivanchenko, S., Röcker, C., Oswald, F., Wiedenmann, J. and Nienhaus, G.U. (2005) Targeted green-red photoconversion of EosFP, a fluorescent marker protein. Journal of Biological Physics, 31 (3-4), 249-259. (doi:10.1007/s10867-005-0174-z).

Abstract

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.

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Contributors

Author: J. Wiedenmann

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