Structural Organization of Proteins on the Oxidizing Side of Photosystem II

本项目描述了蓝细菌Synechocystis 6803中携带外源性环区CPal病变的突变体的产生和初步特征。CPal（由psbB基因编码）是一种内在的膜蛋白，是蓝藻和高等植物光合机构的一个组成部分，似乎是光系统II中光合氧气进化所必需的。该项目的目标是确定该蛋白的大而不寻常的外部环区域的功能。这个区域似乎与外在的33 kDa蛋白和可能的无机辅因子锰、氯或钙相互作用，所有这些都是氧气释放所必需的。此外，CPal似乎是光系统II的叶绿素-a天线。外源性环区可能与其他触角蛋白和/或光系统II反应中心相互作用。体外诱变（饱和诱变和寡核苷酸介导的定点诱变）将用于在外源性环区产生突变。这些突变将被重新引入聚胞菌（连同密切相关的抗生素耐药性标记物），突变体将通过抗生素耐药性筛选和光自养生长丧失相结合的方法分离出来。此外，筛选抗生素耐药性和光自养生长损失。此外，还将进行温度敏感突变的筛选。这些突变将通过检查它们的电子传递活性（77oK）荧光发射光谱、光系统ii富集制剂的锰和钙含量、光合膜蛋白组成以及它们稳定锰所需的氯化物来表征。

This project describes the production and preliminary characterization of mutants bearing lesions in the extrinsic loop region of CPal in the cyanobacterium Synechocystis 6803. CPal (encoded by the psbB gene) is an intrinsic membrane protein which is an integral component of the photosynthetic apparatus of cyanobacteria and higher plants and appears to be required for photosynthetic oxygen evolution in Photosystem II. The goal of this project is to determine the function of the large and unusual extrinsic loop region of this protein. This region appears to interact with the extrinsic 33 kDa protein and possibly the inorganic cofactors manganese, chloride, or calcium, of all of which are required for oxygen evolution. Additionally, CPal appears to act as a chlorophyll-a antenna for Photosystem II. The extrinsic loop region may interact with other antennae proteins and/or the Photosystem II reaction center. In vitro mutagenesis (saturation and oligonucleotide-mediated site- directed) will be used to produce mutations in the extrinsic loop region. These mutations will be re-introduced into Synechocystis (along with a closely linked antibiotic resistance marker), and mutants will be isolated by a combination of screening for antibiotic resistance and loss of photoautotrophic growth. Additionally, screening for antibiotic resistance and loss of photoautotrophic growth. Additionally, screening for temperature sensitive mutations will be performed. These mutations will then be characterized by examination of their electron transport activity 77oK fluorescence emission spectra, manganese and calcium content of Photosystem II-enriched preparations, photosynthetic membrane protein composition, and their chloride requirements for the stabilization of manganese.