Continuous fluorescence assay of phytochrome assembly in vitro.

Incubation of recombinant apophytochrome with the phycobiliprotein chromophore precursor phycoerythrobilin produces a covalent adduct that exhibits a fluorescence excitation maximum at 576 nm and an emission maximum at 586 nm. Using these fluorescence parameters, we have developed a kinetic assay for quantitative analysis of the assembly of the plant photoreceptor phytochrome in real time. Kinetic measurements performed with different phycoerythrobilin concentrations confirm that bilin attachment to apophytochrome involves two steps, an initial formation of a reversible non-covalent complex followed by thioether bond formation. The kinetic constants for both steps of phycoerythrobilin attachment to apophytochrome were estimated with this assay. Methodology for determining the kinetic constants for the assembly of both the natural phytochrome chromophore precursor, phytochromobilin, and the analog phycocyanobilin is also described. Since the latter two bilins yield covalent, nonfluorescent adducts with apophytochrome, their co-incubation with phycoerythrobilin reduces the rate of formation of the fluorescent phycoerythrobilin adduct in an irreversible, competitive manner. Competition experiments were also performed with biliverdin, a structurally related bilin which does not form a covalent adduct with apophytochrome. Such measurements show that biliverdin reversibly binds to apophytochrome with a submicromolar binding constant, an affinity which is very similar to that of phytochromobilin. The utility of this fluorescence assay for identification of novel inhibitors of phytochrome assembly and for characterization of the structural features of both bilin and apophytochrome necessary for photoreceptor assembly is discussed.