| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2006: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2005: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2004: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2003: ¥5,300,000 (Direct Cost: ¥5,300,000)
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| Research Abstract |
Phytochromes are photoreceptors existing in plants and carry a bilin chromophore, which is covalently attached to the protein. Phytochromes behave as photoreversible molecular switches between physiologically inactive red light-absorbing Pr and physiologically active far-red light-absorbing Pfr. This "red-far-red lights photoreversible reaction" plays important role to sense light information in the environment and related to various processes such as development, growth, and differentiation of plants. In the present research project on phytochromes, we established the efficient methods for the preparation of bilin chromophores and their sterically locked derivatives, which were assembled with apoproteins of phytochromes. The following are the results obtained by synthetic organic approach toward elucidation of the structure and function of phytochromes
1. Many bacterial phytochromes carry biliverdin (BV) as natural chromophore, which is coupled in a different manner to the protein. In
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phytochrome Agpl of Agrobacterium tumefaciens, BV is covalently attached to a cysteine residue close to the N-terminus (position 20). By testing different natural and synthetic BV derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment.
2. Total syntheses of BV derivatives with sterically locked AB-or CD-ring component were accomplished via new and efficient methods towards elucidation of the stereochemistry of the chromophore in phytochromes. The chromophores were tested for assembly with Agrobacterium phytochrome Agpl and Agp2, which incorporate BV as natural chromophore. It was found that the chromophore of Agpl adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form, and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agpl. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.
3. The N-terminal photosensory module of Agrobacterium phytochrome Agpl, Agp1-M15, was assembled with a sterically locked synthetic BV-derivative 15Za to give crystals of 15Za-Agpl-M15, which diffract to 3.4 A resolution and belong to the tetragonal space group 1422 with unit cell dimensions a=b=174 A, c=80 A.
4. A new type of affinity chromatography was investigated to establish an efficient method for the isolation of plant phytochromes by attaching synthetic chromophores as a ligand on carrier resin.
5. Chromophores doubly locked at AB-and CD-rings were synthesized to determine the stereochemistry of AB-ring component of Pfr and also toward analysis of the detailed mechanism of photoisomerization of Agpl and signal transduction.
6. To exploit an efficient method for the preparation of optically active phytochrome chromophores, new highly efficient and enantioselective reactions were developed utilizing tartaric acid esters as a chiral auxiliary. Furthermore new insights were obtained regarding "syn-effect". Less
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