USE OF MUTANTS TO STUDY CYANOBACTERIAL MEMBRANE PROTEINS

该项目的长期目标是开发和利用 基因工具来研究膜的生物起源和结构， 结合蛋白质复合物。 的主题 直接的研究是光合作用膜， 特别地，两种完整的蛋白质复合物，光系统I（PSI）和 光系统（PSII）。 该建筑群具有结构性， 与细菌反应中心的功能相似性，第一个 膜蛋白复合物，其X射线晶体数据已成为 available. 这使得人们可以假设 PSII中不同的蛋白质结构域，并使用遗传方法， 检查这些假设。 单细胞的，可转化的 蓝细菌，集胞藻6803将用于本项目， 蛋白质-蛋白质以及蛋白质-辅因子的遗传分析 PSII中的相互作用。 PSI复合物也被结晶化了 在过去的一年中，结构数据预计将获得 很快 然而，没有一个已经表征的蓝藻 系统（包括集胞藻）可用于遗传分析 的PSI。 本项目的拟议研究将为 第一次，遗传工具来研究PSI复合体在原核生物中， 微生物，念珠藻29150。 了解结构和装配 这两个完整的膜结合蛋白复合物将是一个 对所有蛋白质的一般知识的重要贡献- 类似的复合物。 这些研究还将完善 今后工作中将采用的技术和概念， 膜蛋白复合物在生物医学上很重要。 生物化学、遗传学、生物物理学和重组学的结合 DNA技术将用于研究形成的调控 以及PSI和PSII复合物的结构。 的 本建议的直接目标是：1）诱变 psbA基因，编码一个快速翻转的“D1”蛋白， 集胞藻6803的PSII复合体; 2）PSII复合体的克隆和诱变 Nostoc 29150的PSI基因。 由此产生的突变体将 分析以检查（a）一种蛋白质中的一种蛋白质是否突变 复合物允许复合物的其余部分在 膜和（B）复合物的哪些功能活性 被蛋白质中的特定突变所抑制。 我们亦会 进行随机诱变实验以鉴定新的结构 以及在这些蛋白质中起重要作用的调节位点 配合物 将开发膜下制剂， 这些突变体的物理化学分析。

The long-term objective of this project is to develop and utilize genetic tools to study the biogenesis and architecture of membrane- bound protein complexes in cyanobacteria. The subject of the immediate investigation is the photosynthetic membrane and in particular, two integral protein complexes, Photosystem I (PSI) and Photosystem (PSII). The complex has structural as well as functional similarities with a bacterial reaction center, the first membrane protein complex whose xray crystal data have become available. This allows one to hypothesize about putative roles of different protein domains in PSII and use genetic approaches to examine these hypotheses. The unicellular, transformable cyanobacterium, Synechocystis 6803 will be used in this project for genetic analysis of protein-protein as well as protein-cofactor interactions in PSII. The PSI complex has also been crystallized in the past year and structural data are expected to be available soon. However, none of the already characterized cyanobacterial systems (including Synechocystis) can be used for genetic analysis of PSI. The proposed studies in this project will provide, for the first time, genetic tools to study the PSI complex in a prokaryotic organism, Nostoc 29150. Understanding the structure and assembly of these two integral membrane-bound protein complexes will be an important contribution to the general knowledge about all protein- complexes of similar kind. These studies will also refine techniques and concepts that will be employed in future work on membrane-protein complexes that are biomedically important. A combination of biochemical, genetic, biophysical and recombinant DNA techniques will be used to study the regulation of formation as well as the architecture of the PSI and PSII complexes. The immediate objectives in this proposal will be: 1) mutagenesis of the psbA gene, encoding a rapidly turned over 'D1' protein of the PSII complex in Synechocystis 6803; 2) cloning and mutagenesis of PSI genes from Nostoc 29150. The resultant mutants will then be analyzed to examine (a) whether mutation in one protein in a complex allows the rest of the complex to be assembled in the membrane and (b) which functional activities of a complex are inhibited by a specific mutation in a protein. We shall also perform random mutagenesis experiments to identify new structural and regulatory loci that play important roles in these protein complexes. Submembrane preparations will be developed for physicochemical analyses of these mutants.