Preparation of single crystal of phytochrome and its preliminary structural analysis by X-ray crystallography

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Phytochrome is a chromoprotein in green plants, which serves as a photoreceptor for a variety of morphogenetic responses. In order to elucidate its three-dimensional structure. crystallographic study has been started. First, conditions for crystallization of large (114kDa, exist in a dimer) phytochrome was investigated systematically. Partly crystallized blocks (0.5mm) were obtained using ammonium sulfate as precipitant, however, preparation of large single crystals has not been successful yet. In stead, crystallization of small (59kDa, chromophoric domain) phytochrome was started, which seems promissing judging from the results obtained so far, and is to be continued.In parallel to the above research, molecular structure of the chromophore was studied using resonance Raman scattering (Tokutomi et al., 1990, FEBS Lett., Mizutani et al., Bioahem., 1991), which showed a different protoated structure between the red- and far redabsorbing forms. A model for molecular struture of the chromophore was constructed using these new informations (Toktutomi et al., 1992, Photochem. Photobiol.), indicated elongated molecular structure. Its orientation in the dimeric phytochrome molecule, proposed based on small-angle X-ray scattering (Nakasako et al., 1990, Photochem. photobiol.), was determined based on linear dichroism of oriented molecules (Toktutomiet al., i992, Photoahem. Photobiol.), which shows that the chromophore is well contained in a flat chromophoric domain of the dimeric molecule. These results can supply useful informations to the crystallographic anlysis of phytochrome.