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Assembly of Photosynthetic Apparatus on Cyanobacteria

蓝藻光合装置的组装

基本信息

批准号：
09044209
负责人：
HISABORI Toru
金额：
$ 3.84万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044209/
关键词：
photosynthetic apparatus GroEL ES system ATP synthase γsubunit εsubunit complex

项目摘要

On the biological membranes, many different protein complexes are functioning and they have strong relations each other. Recent progress of the genome project and the development of the related molecular biology technique revealed that the interaction among these protein complexes are very intimate not only on the function but also on the biogenesis of them. However, it is not understood very well which kind of interaction control the total function of the multiple complexes and the assembly of the machinery. Here, we tried to clarify the network of the multiple protein complexes on the biological membranes. For this research we focused on the photosynthetic apparatus on the thylakoid membrane. In 1996, the whole genome sequences of the cyanobacteria, the potential origin of the chloroplast of higher plants, were revealed by the researchers of Kazusa DNA research laboratory Japan and the information of the proteins at the amino acid level for the whole machinery of the photosynthetic c … More omplexes of this bacteria has become available. We here adopted this advantage for our study.Additionally, we chose FィイD20ィエD2FィイD21ィエD2 ATP synthase, one of the most complex apparatus on the thylakoid membrane as a research target for the study of the assembly of the molecular machinery.1. The evaluation of the role of GroEL/ES system of cyanobacteria for the protein complex assembly and the protein expression in E. coliGroEL/ES system facilitates folding of non-native proteins in vivo and in vitro. The most well-studied GroEL/ES complex is the one from Escherichia coli. The crystal structure of this complex is already reported. The complex has a cylindrical cavity which can accept the unfolded peptides. The Similar protein complex was found in Synechocystis PCC6803 cell (Plant Moi. Biol. (1992) 18, 327-336). The larger subunit of the complex (HSP64) had the homology with GroEL and was assigned as GroEL- related chaperonin. As there are some reports that the bacterial GroEL/ES system could assist the well expression of the introduced proteins, we here examined whether the GroEL/ES complex from Synechocystis can assist the expression of the certain protein of cyanobacteria in E. coli cell.For this purpose, the genes for GroEL, GroEL2 (GroEL homolog), and GroES were obtained from total DNA of Synechocystis PCC6803 by PCR method and the expression vectors for each of them were constructed. As we already had several proteins from Synechocystis cell, of which expression in E. coli was very difficult, we here tried the co-expression of these proteins with the GroEL/ES system of cyanobacteria. Interestingly, although most of these proteins were expressed as inclusion body in E. coil cell, the expression of the one of these proteins from cyanobacteria was strongly affected by the co-expression of cyanobacterial GroEL/ES system, thus giving the soluble protein.2. On the assembly of the ATP synthaseTo understand the functional biogenesis of the chloroplast ATP synhtase, we here studied the interaction between γ subunit or ε subunit and αィイD23ィエD2βィイD23ィエD2 core complex. For this purpose, a couple of mutant γ subunit were expressed as recombinant proteins. To asses the effects of these mutations, the ATPase active chimeric complex was successfully reconstituted with the recombinant γ subunit and the α and β subunits from thermophilic bacteria PS3. By using this system, we could assign several important region for the regulation of the enzyme activity. In case of ε subunit, the authentic αィイD23ィエD2βィイD23ィエD2γcomplex prepared from chloroplast coupling factor ATPase were used for the study of the inhibition effect. We could find several important charged residues on the putative α-helix structure (predicted from the homology with bacterial ε subunit) for the function.Furthermore, we investigated the regulatory mechanism of chloroplast ATP synthase in light of the interaction between the enzyme and the regulator protein, thioredoxin. From the study, we found the novel interaction between them which will raise the conformational change of the target region. This thioredoxin-induced conformational change must be important for the efficient activation of the enzyme. Less

在生物膜上，许多不同的蛋白质复合物在起作用，它们之间有很强的联系。近年来基因组计划的进展和相关分子生物学技术的发展表明，这些蛋白质复合物不仅在功能上相互作用密切，而且在生物发生过程中也有着密切的相互作用。然而，究竟是哪一种相互作用控制了多个复合物的总体功能和机械的组装，目前还不清楚。在这里，我们试图澄清多种蛋白质复合物在生物膜上的网络。本研究重点研究了类囊体膜上的光合装置。1996年，日本Kazusa DNA研究实验室的研究人员揭示了高等植物叶绿体的潜在起源蓝藻的全基因组序列，并在氨基酸水平上揭示了整个光合作用机制的蛋白质信息。我们在这里利用这一优势进行研究。此外，我们选择了类囊体膜上最复杂的装置之一的ATP合酶F _ (20 _ (20) _ (21) _ (21) _ (D2) ATP合酶作为研究对象，对其分子机制的组装进行了研究。研究蓝藻GroEL/ES系统在蛋白质复合物组装中的作用，以及e.c oli igroel /ES系统中蛋白质的表达对体内和体外非天然蛋白折叠的促进作用。研究最充分的GroEL/ES复合体来自大肠杆菌。这种复合物的晶体结构已被报道过。该配合物具有圆柱形空腔，可接受未折叠的肽。相似的蛋白复合物在聚囊藻PCC6803细胞（Plant Moi）中被发现。医学杂志。（1992） 18, 327-336)。该复合物的较大亚基（HSP64）与GroEL具有同源性，并被指定为GroEL相关伴侣蛋白。由于有报道称细菌GroEL/ES系统可以帮助导入蛋白的良好表达，我们在这里研究了Synechocystis的GroEL/ES复合体是否可以帮助大肠杆菌细胞中蓝藻的某些蛋白的表达。为此，采用PCR方法从聚囊藻PCC6803的总DNA中获得GroEL、GroEL2 （GroEL同源物）和GroES基因，并构建各自的表达载体。由于我们已经从聚囊藻细胞中获得了几种蛋白，这些蛋白在大肠杆菌中表达非常困难，我们在这里尝试将这些蛋白与蓝藻的GroEL/ES系统共表达。有趣的是，虽然这些蛋白大部分在E. coil细胞中以包涵体形式表达，但其中一种蛋白在蓝藻中的表达受到蓝藻GroEL/ES系统共表达的强烈影响，从而获得可溶性蛋白2。组装的ATP synthaseTo了解叶绿体的功能生物起源ATP synhtase,我们研究了γ亚基之间的相互作用或ε单元和αィイc15ィエD2βィイc15ィエD2核心复杂。为此，将一对突变体γ亚基表达为重组蛋白。为了评估这些突变的影响，我们成功地用重组的γ亚基和来自嗜热细菌PS3的α和β亚基重组了ATPase活性嵌合物。利用该系统，我们可以确定几个调控酶活性的重要区域。对于ε亚基，采用由叶绿体偶联因子ATPase制备的正品α γ γ γ配合物来研究其抑制作用。我们可以在推测的α-螺旋结构（从与细菌ε亚基的同源性预测）上找到几个重要的带电残基。此外，我们还从ATP合成酶与调节蛋白硫氧还蛋白的相互作用角度探讨了叶绿体ATP合成酶的调控机制。从研究中，我们发现了它们之间新的相互作用，这种相互作用会引起靶区的构象变化。这种硫氧还蛋白诱导的构象变化对于酶的有效激活至关重要。少