THE PHYTOFLUORS - A NEW CLASS OF FLUORESCENT PROTEIN PROBES

Background: Biologically compatible fluorescent protein probes, partic ularly the self-assembling green fluorescent protein (GFP) from the je llyfish Aequorea victoria, have revolutionized research in cell, molec ular and developmental biology because they allow visualization of bio chemical events in living cells. Additional fluorescent proteins that could be reconstituted in vivo while extending the useful wavelength r ange towards the orange and red regions of the light spectrum would in crease the range of applications currently available with fluorescent protein probes. Results: Intensely orange fluorescent adducts, which w e designate phytofluors, are spontaneously formed upon incubation of r ecombinant plant phytochrome apoproteins with phycoerythrobilin, the l inear tetrapyrrole precursor of the phycoerythrin chromophore. Phytofl uors have large molar absorption coefficients, fluorescence quantum yi elds greater than 0.7, excellent photostability, stability over a wide range of pH, and can be reconstituted in living plant cells. Conclusi ons: The phytofluors constitute a new class of fluorophore that can po tentially 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 synthes is are expected to afford new phytofluors with fluorescence excitation and emission spectra spanning the visible to near-infrared light spec trum.