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Synthetic Approach toward Elucidation of the Structure and Function of Phytochromes

阐明光敏色素结构和功能的综合方法

基本信息

批准号：
15350021
负责人：
INOMATA Katsuhiko
金额：
$ 9.47万
依托单位：
KANAZAWA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

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 phytochromes1. Many bacterial phytochromes carry biliverdin (BV) as natural chromophore, which is coupled in a different manner to the protein. In … More 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

光敏色素是存在于植物中的光感受器，并携带胆色素发色团，其共价连接到蛋白质上。光敏色素表现为生理上无活性的红光吸收Pr和生理上有活性的远红光吸收Pfr之间的光可逆分子开关。这种“红-远-红光光可逆反应”在感知环境中的光信息以及与植物的发育、生长、分化等过程相关的光信息方面起着重要作用。本课题以光敏色素为研究对象，建立了高效的胆色素发色团及其空间锁定衍生物的制备方法，并将其与光敏色素脱辅基蛋白组装在一起。以下是通过有机合成方法阐明光敏色素1的结构和功能所获得的结果。许多细菌光敏色素携带胆绿素（BV）作为天然发色团，其以不同的方式与蛋白质偶联。在 ...更多信息在根癌土壤杆菌的光敏色素Agpl中，BV共价连接到靠近N-末端的半胱氨酸残基（位置20）。通过测试不同的天然和合成BV衍生物，发现环A乙烯基侧链用于发色团的连接.通过新的有效的方法，实现了具有空间锁AB环或CD环组分的BV衍生物的全合成，以阐明光敏色素中发色团的立体化学。测试发色团与农杆菌光敏色素Agpl和Agp 2的组装，所述农杆菌光敏色素Agpl和Agp 2并入BV作为天然发色团。结果表明，Agpl的Pr型发色团为C_（15）= C_（16）Z构型和C_（14）-C_（15）反构象，Pfr型发色团为C_（15）= C_（16）E构型和C_（14）-C_（15）反构象。通过尺寸排阻色谱和组氨酸激酶活性分析所有发色团加合物以探测蛋白质构象。在这两种情况下，15 Za加合物的行为与Pr相似，15 Ea加合物的行为与Agpl的Pfr形式相似。因此，用锁定的15 Ea衍生物取代天然发色团可以在黑暗中以Pfr形式产生光敏色素全蛋白。通过这种方式，可以在体内研究Pfr的生理作用，并将其与Pr/Pfr循环和其他光效应分开.将农杆菌光敏色素Agpl的N-末端光敏模块Agpl-M15与空间锁定的合成BV-衍生物15 Za组装，得到15 Za-Agpl-M15的晶体，其分辨率为3.4 A，属于四元空间群1422，晶胞尺寸a=B=174 A，c=80 A。研究了一种新型的亲和层析方法，将人工合成的光敏色素作为配基连接到载体树脂上，建立了一种高效分离植物光敏色素的方法.合成了AB环和CD环双锁定的生色团，以确定Pfr AB环组分的立体化学，并分析Agpl光异构化和信号转导的详细机制.为了探索一种制备光学活性光敏色素发色团的有效方法，利用酒石酸酯作为手性助剂，发展了一种新的高效的对映选择性反应。此外，新的见解获得了关于“同步效应”。少

项目成果

期刊论文数量（71）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

"Syn-Effect" in the Conversion of (E)-α,β-Unsaturated Esters to the Corresponding β,γ-Unsaturated Esters

(E)-α,β-不饱和酯转化为相应的β,γ-不饱和酯的“顺式效应”

DOI：
发表时间：
2003
期刊：
Chem. Lett. 32・8
影响因子：
0
作者：
Mostafa A.;S.Hammam;Samar Kumar Guha;猪股勝彦;Mostafa A.S. Hammam;Samar Kumar Guha;M.A.S.Hammam;S.K.Guha;猪股勝彦;Tilman Lamparter;Takanori Mizutani;Xia Ding;Yutaka Ukaji;Samar Kumar Guha
通讯作者：
Samar Kumar Guha

Biliverdin binds covalently to Agrobacterium phytochrome Agp1 via its ring a vinyl side chain