MOLECULAR BIOLOGY AND BIOCHEMISTRY OF PHOTOSYSTEM II

这个项目的长期目标是使用分子工具 生物学和生物化学，以了解各种基因和它们的作用 调节色素-蛋白质复合体功能的产物， 光系统EL一个更直接的目标是了解功能 一些非常小的蛋白质(10 KDa)在该多蛋白中的作用 我们的实验生物体是单细胞的，可变形的 蓝藻，聚球藻6803。在过去，我们用‘反向’ 遗传学的方法在许多基因中创造特定的靶向突变 聚球藻6803各种PSII蛋白的编码基因。我们现在 计划使用类似的方法在基因中创建定点突变 编码这种蛋白复合体的小蛋白PSII复合体具有 与反应中心在结构和功能上的相似性， 第一个膜结合型多蛋白复合体，其X射线晶体数据 变得有空。然而，反应中心复合体不包含 任何一种小的蛋白质成分。有趣的是，这样的小蛋白质 许多膜蛋白复合体的普遍成分，如细胞色素 氧化物酶。这种小蛋白的结构和/或功能作用 然而，人们对此并不清楚。E本项目中的拟议研究 从而，拓宽了我们对这种小的功能的一般认识 所有蛋白质中的蛋白质--类似种类的复合体。这些研究还将 改进可能在今后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复合体。 生化、遗传和重组DNA技术的结合 将被用来研究不同突变位点定向的影响 在此提案期间创建的突变体。此外，我们还将 分离随机诱变的PSII缺陷突变体。特别的 有趣的是使用转座子元件Tn5tac 1来创建 条件性(IPTG，一种乳糖类似物，依赖)突变体 他们的PSII活动。使用这种多功能的基因工具和便捷的 聚球藻6803转化系统将帮助我们鉴定 结构和更重要的是，影响经济增长的监管因素 PSII复合体的生物发生、稳定性和功能。

The long term objective of this project is to use tools of molecular biology and biochemistry to understand the roles of various genes and their products in the regulation of function of a pigment - protein complex, Photosystem EL A more immediate objective is to understand the functional role of a number of very small proteins (< 10 kDa) in this multiprotein complex Our experimental organism is a unicellular, transformable cyanobacterium, Synechocystis 6803. In the past, we have used 'reverse genetics' approaches to create specific targeted mutations in a number of genes that encode various PSII proteins of Synechocystis 6803. We now plan to use similar approaches to create site directed mutants in genes encoding small proteins of this protein complex The PSII complex has structural as well as functional similarities with a reaction center, the first membrane bound multiprotein complex whose Xray crystal data have become available. However, the reaction center complex does not contain any small protein component. Interestingly, such small proteins are ubiquitous components of many membrane protein complexes, e.g., cytochrome oxidase. The structural and/or functional roles of such small proteins are, however, not clearly understood. e proposed studies in this project will thus, broaden our general understanding of the function of such small proteins in all protein-complexes of similar kind. These studies will also refine techniques and concepts that may be employed in future work on membrane protein complexes that are biomedically important. A combination of biochemical, genetic and recombinant DNA techniques will be used to study the effects various mutations in the site directed mutants created during this proposal period. In addition, we will to isolate randomly mutagenized PSII deficient mutants. Of particular interest will be the use of a transposon element, Tn5tac 1, to create conditional (IPTG, a lactose analog, -dependent) mutants that impaired in their PSII activities. Use of such versatile genetic tools and the facile transformation system Synechocystis 6803 will help us in identifying novel structural and, more important, regulatory loci that influence the biogenesis, stability, as well as function, of the PSII complex.