Targeted Green-Red Photoconversion of EosFP, a Fluorescent Marker Protein.
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.
Department of Biophysics, University of Ulm, 89069 Ulm, Germany.
Sergey
S
Ivanchenko
Carlheinz
C
Röcker
Franz
F
Oswald
Jörg
J
Wiedenmann
G Ulrich
GU
Nienhaus
fluorescence spectroscopy; fluorescent protein; fusion protein; photoconversion; two-photon excitation