EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion

Abstract

A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at Ϸ390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Fö rster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

“…In an earlier paper (27), we have reported that H-NS appears as a few large and discrete clusters in cells when fused to mEos2, a previously reported monomeric version of the Eos fluorescent protein (14,16). We also observed similar results when H-NS was fused to PAmCherry (27), another previously reported monomeric PAFP.…”

“…Here, we compare four properties of PAFPs that are critical for superresolution imaging and report two new PAFPs that exhibit excellent performance in all four properties. (14), mEos3.2 (15), tdEos (16), mKikGR (17), PAmCherry (18), PAtagRFP (19), mMaple (20), PSCFP2 (13,21), Dronpa (22), and mGeosM (23). From this screen, we found that none of these PAFPs was simultaneously optimal in all four criteria described above.…”

Fluorescence nanoscopy. Methods and applications

“…Photochromatism usually involves a two-step photooxidation process that causes cleavage of the polypeptide backbone (e.g. PSmOrange and EosFP [138,144]). Photoswitching involves cis-trans isomerisation of the chromophore accompanied by a change of its protonation state (the anionic state being the fluorescent state and the protonated the nonfluorescent-interestingly, the chromophore can be fluorescent in both the cis and the trans conformations as long as the protein scaffold keeps it in a planar conformation) [145][146][147].…”

Fluorescent proteins for live cell imaging: Opportunities, limitations, and challenges

“…In another group of FPs, the p-HBI chromophore can be converted irreversibly from a green to a red fluorescent state by a photochemical modification of the peptide backbone. In EosFP, Kaede and several other anthozoan FPs and variants, irradiation with 400 nm results in a cleavage of the peptide backbone between N a and C a of the first chromophore-forming residue histidine (63)(64)(65)(66)(67). Thereby, the conjugated p-electron system is extended in the imidazole sidechain of histidine.…”

“…Great experimental opportunities arise from the diversity of fluorophores for multicolor labeling of proteins, cellular compartments or cells as well as for novel sensors based on fluorescence resonance energy transfer between FPs of different colors (8,39,57,66). Currently, at least five differently colored FPs can be imaged in parallel (8,59).…”