USE OF MUTANTS TO STUDY CYANOBACTERIAL MEMBRANE PROTEINS

该项目的长期目标是开发和利用 研究膜的生物发生和结构的遗传工具- 蓝藻中的结合蛋白复合体。的主题是 直接的研究是光合膜和在 特别是，两个完整的蛋白质复合体，光系统I(PSI)和 光系统(PSII)。该建筑群既有结构上的，也有 与细菌反应中心的功能相似，第一 膜蛋白复合体的X射线晶体数据已成为 可用。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 检验一下这些假设。单细胞的，可变形的 蓝藻，聚球藻6803将用于本项目 蛋白质-蛋白质和蛋白质辅因子的遗传分析 PSII中的相互作用。PSI复合体也已经结晶 在过去的一年里，结构性数据预计将可用 很快。然而，没有一种已经具有特征的蓝藻 系统(包括聚球藻)可用于遗传分析 PSI。这项计划的拟议研究将为 首次用遗传学工具研究原核生物中的PSI复合体 有机体，发酵剂29150。了解结构和组装 这两个完整的膜结合蛋白复合体中将有一个 对所有蛋白质的常识做出了重要贡献- 类似类型的复合体。这些研究还将完善 将在未来的工作中使用的技术和概念 具有重要生物医学意义的膜-蛋白质复合体。 生化、遗传、生物物理和重组的结合 DNA技术将被用来研究形成的规则 以及PSI和PSII复合体的架构。这个 该提案的近期目标将是：1)诱变 PSBA基因编码一种快速翻转的‘d1’蛋白。 集胞藻6803中PSII复合体的克隆及诱变 发菜29150的psi基因。由此产生的突变体将会是 分析以检查(A)一种蛋白质中的突变是否 Complex允许将Complex的其余部分组装在 膜和(B)络合物的哪些功能活性 被蛋白质中的特定突变抑制的。我们还将 进行随机诱变实验以确定新的结构 以及在这些蛋白质中发挥重要作用的调控基因座 复合体。亚膜制剂将被开发用于 对这些突变体进行了物理化学分析。

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.