Structural Organization of Proteins on the Oxidizing Side of Photosystem II

本项目描述了蓝藻聚球藻6803中CPal外环区域携带损伤的突变体的产生和初步特征。CPal(由psbB基因编码)是蓝藻和高等植物光合作用机构的组成部分，在光系统II中似乎是光合作用放氧所必需的内源性膜蛋白。本项目的目的是确定该蛋白的大而不寻常的外环区的功能。这一区域似乎与外源性33 kDa蛋白相互作用，可能还与无机辅因子锰、氯或钙相互作用，所有这些都是放氧所必需的。此外，CPal似乎是光系统II的叶绿素-a天线。外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。在体外诱变(饱和和寡核苷酸介导的定点定向)将被用来在外环区域产生突变。这些突变将被重新引入聚球藻(连同一个紧密相连的抗生素耐药性标记)，并通过筛选抗生素耐药性和失去光自养生长来分离突变株。此外，对抗生素抗性和光自养生长丧失进行筛选。此外，还将进行温度敏感突变的筛查。然后将通过检查它们的电子传递活性、77oK荧光发射光谱、富含光系统II的制剂的锰和钙含量、光合膜蛋白组成以及它们对稳定锰的氯化物需求来表征这些突变。

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.