The phytofluors: a new class of fluorescent protein probes

AbstractBackground: Biologically compatible fluorescent protein probes, particularly the self-assembling green fluorescent protein (GFP) from the jellyfish Aequorea victoria, have revolutionized research in cell, molecular and developmental biology because they allow visualization of biochemical events in living cells. Additional fluorescent proteins that could be reconstituted in vivo while extending the useful wavelength range towards the orange and red regions of the light spectrum would increase the range of applications currently available with fluorescent protein probes.Results: Intensely orange fluorescent adducts, which we designate phytofluors, are spontaneously formed upon incubation of recombinant plant phytochrome apoproteins with phycoerythrobilin, the linear tetrapyrrole precursor of the phycoerythrin chromophore. Phytofluors have large molar absorption coefficients, fluorescence quantum yields greater than 0.7, excellent photostability, stability over a wide range of pH, and can be reconstituted in living plant cells.Conclusions: The phytofluors constitute a new class of fluorophore that can potentially be produced upon bilin uptake by any living cell expressing an apophytochrome cDNA. Mutagenesis of the phytochrome apoprotein and/or alteration of the linear tetrapyrrole precursor by chemical synthesis are expected to afford new phytofluors with fluorescence excitation and emission spectra spanning the visible to near-infrared light spectrum