Assembly of Photosynthetic Apparatus on Cyanobacteria

在生物膜上，许多不同的蛋白质复合体是功能性的，它们彼此之间有很强的关系。基因组项目的最近进展和相关分子生物学技术的发展表明，这些蛋白质复合体之间的相互作用非常亲密，并不只是在功能上，但也是在它们的生物起源上。However，这并不是很好地理解这种交互控制的多个复杂性的总功能和机器的组装。在这里，我们试图澄清生物膜上的多蛋白质复合体网络。在这项研究中，我们集中精力研究了甲状腺状膜上的光电合成装置。在1996年，《青细菌的整个基因组序列、高植物的氯plast的潜在起源》由日本Kazusa DNA研究实验室研究人员公布，并提供了关于光合成c整个机器的氨基酸水平的蛋白质信息。 ... More 此细菌的复杂性已成为可用的。We here adopted this advantage for our study。Additionally， we chose FイイD20イD2FイD21イD2 ATP synthase， one of the most complex apparatus on the thylakoid membrane as a res？target for the study of the assembly of the molecular machinery. 1。GroEL/ES对蛋白质复合体组件和蛋白质表达的作用的评估。coliGroEL/ES系统的设施折叠在体内和体外的非原生蛋白质。研究最好的Groel/ES复合体是大肠杆菌的一个。该复合体的水晶结构已被报道。这个复合体有一个圆柱形洞穴，它可以接受未折叠的肽。在Synechocystis PCC 6803细胞中发现的类似蛋白复合体（Plant Moi）。1992年，第18， 327-336段。复杂的大子单元（HSP 64）具有GroEL和was assigned as GroEL-related chaperonin。正如有一些关于细菌GroEL/ES系统可以很好地帮助诱导蛋白质的表达的报道，我们在这里研究了从Synechocystis中的GroEL/ES复合体可以帮助解释蓝细菌中某些蛋白质的表达的报道。大肠杆菌细胞，用于这种目的，用于GroEL、GroEL 2（GroEL同源物）的基因和GroES从综合细胞系PCC 6803的总DNA中获得，并用于构建它们的每个表达载体。正如我们已经从Synechocystis细胞中发现了一些蛋白质，在E.中的表达。大肠杆菌是非常困难的，我们在这里用GroEL/ES系统研究了这些蛋白质的共同表达。有趣的是，这些蛋白质中的大多数也被表达为E.。Coil Cell，其中一种蛋白质从Cyanobacteria中受到强烈影响的表达，即Cyanobacteria GroEL/ES系统的共同表达，从而赋予了溶解蛋白质。On the assembly of the ATP synthaseTo understand the functional biogenesis of the chloroplast ATP synhtase， we here studied the interaction between·由于这个目的，一对突变γ亚单位被表达为重组蛋白。为了分析这些突变的影响，ATPase活性化学复合体成功地与热细菌PS3的重组γ亚单位和α和β亚单位重新构建。由于使用这个系统，我们可以为酶活性的规定指定几个重要的区域。In case of？subunit， the authentic？D23？complex prepared from chloroplast coupling factor ATPase were used for the study of the inhibition effect。我们可以为函数找到几个重要的电荷替代方案（由细菌ε亚单位预测的同源性）。Furthermore，我们研究了在酶和调节蛋白质之间相互作用的光照下氯铂ATP合成酶的调节机制，硫唑酮。从研究中，我们在目标地区之间找到了新的互动，这些互动将引起正常的变化。这种由三氟代辛诱导的适应性变化对于酶的有效激活是至关重要的。Less（低）

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