Structural Organization of Proteins on the Oxidizing Side of Photosystem II

本项目介绍了生产和初步 在外环中具有损伤的突变体的表征 蓝细菌集胞藻6803中的CPal区域。 CPal （由psbB基因编码）是一种内在膜蛋白， 是光合器官的组成部分， 蓝藻和高等植物，似乎需要 光系统II中的光合放氧。 的目标 该项目是为了确定大型和 这种蛋白质的不寻常的外部环区域。 这一地区 似乎与外源性33 kDa蛋白相互作用， 可能的无机辅因子锰、氯或钙， 这些都是氧气释放所必需的。 此外，本发明还 CPal似乎起着叶绿素-光系统天线的作用 二. 外部环区域可以与其他天线相互作用 蛋白质和/或光系统II反应中心。 体外 诱变（饱和和阿糖胞苷介导的位点- 定向）将用于在外部环中产生突变 地区 这些突变将被重新引入集胞藻 （沿着紧密连锁的抗生素抗性标记），和 突变体将通过筛选 抗生素抗性和光合自养生长的丧失。 此外，筛选抗生素耐药性和丧失 光自养生长 此外，温度筛选 将进行敏感突变。 这些突变将 然后通过检查它们的电子传递来表征 活性77 oK荧光发射光谱，锰和 富含光系统II的制剂的钙含量， 光合膜蛋白组成及其氯化物 对锰的稳定化要求。

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.