RUI: Mutagenesis Studies to Probe Function of the Large Extrinsic Loop of the CP-43 Apoprotein of Photosystem II
RUI:用于探测光系统 II CP-43 脱辅基蛋白大外在环功能的诱变研究
基本信息
- 批准号:9513795
- 负责人:
- 金额:$ 18万
- 依托单位:
- 依托单位国家:美国
- 项目类别:Standard Grant
- 财政年份:1996
- 资助国家:美国
- 起止时间:1996-03-01 至 2000-02-29
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
9513795 Putnam-Evans This project describes the production and characterization of mutants containing random and site-directed lesions in the large extrinsic loop region of the Photosystem II (PSII) protein CP43. CP43 is an integral thylakoid protein and component of the photosynthetic apparatus of higher plants, algae and cyanobacteria. These mutants will be constructed in the cyanobacterium Synechocystis 6803, a widely used model system for higher plant photosynthesis. CP43 appears to be required for the stable assembly of the PSII complex, and also appears to play a role in photosynthetic oxygen evolution. The goal of this project is to determine the function of the large, lumenally-oriented extrinsic loop of CP43. Deletion of long segments of the loop results in mutants incapable of evolving oxygen. Therefore, it is probable that this region interacts directly with other PSII proteins and inorganic cofactors (manganese, chloride and calcium) that are necessary for oxygen evolution. In addition, CP43 functions as a chlorophyll-a antenna for PSII. The large extrinsic loop may thus interact with other PSII antenna proteins or proteins comprising the PSII reaction center. Mutations will be introduced into the psbC gene encoding CP43 by both random and oligonucleotide-mediated mutagenesis. Mutations will be re-introduced into Synechocystis 6803 along with a closely-linked antibiotic resistance marker. Putative mutants will be screened initially for antibiotic resistance and possible loss of photoautotrophic growth. This will be followed by an in-depth characterization of photosynthetic characteristics including measurements of steady state rates of oxygen evolution, electron transport capability, PSII polypeptide composition, manganese and calcium content and chloride requirement. %%% The Photosystem II (PSII~ complex forms part ofthe photosynthetic apparatus of higher plants, algae and cyanobacteria. This complex catalyzes the light-driven oxidation of water and the reduction of plastoquinon e. Oxygen is given off as a by-product of the PSII reaction. CP43 is one of the protein components of PSII and appears to be required for the stable assembly ofthe PSII complex. It also appears to play a role in both photosynthetic oxygen evolution and the harvesting of light energy. This membrane-bound protein is unusual in that it contains a large, hydrophilic loop which protrudes from the membrane in which is it embedded. Deletion of short stretches of amino acids in the large extrinsic loop result in mutants that cannot evolve oxygen. The goal of this project is to identify specific amino acids involved in the function of the large, lumenally-oriented extrinsic loop of CP43 to further test the hypothesis that this region is necessary for normal PSII activity. This will be accomplished by 1) introducing mutations into the gene encoding CP43 in vitro using the techniques of both random and site-specific mutagenesis, 2) exchanging these mutated genes for their normal counterparts in the blue-green algae Synechocystis 6803 and 3) evaluating the resulting mutant organisms for their ability to assemble a normal PSII complex, evolve oxygen and carry out electron transport. *** ??
9513795 Putnam-Evans该项目描述了在光系统II(PSII)蛋白CP 43的大的外部环区域中含有随机和定点损伤的突变体的产生和表征。CP 43是高等植物、藻类和蓝细菌的光合机构的组成部分。这些突变体将在蓝藻集胞藻6803中构建,集胞藻6803是高等植物光合作用的广泛使用的模型系统。CP 43似乎是所需的PSII复合物的稳定组装,也似乎在光合放氧中发挥作用。本项目的目标是确定CP 43的大的、面向内腔的外部环的功能。缺失环的长片段会导致突变体无法释放氧气。因此,这是可能的,这个区域直接与其他PSII蛋白和无机辅因子(锰,氯和钙),是必要的氧的演变。此外,CP 43作为叶绿素a天线的PSII。因此,大的外在环可以与其他PSII天线蛋白或包含PSII反应中心的蛋白相互作用。将通过随机诱变和多核苷酸介导的诱变将突变引入编码CP 43的psbC基因中。突变将与紧密连锁的抗生素抗性标记一起重新引入集胞藻6803沿着。初步筛选推定的突变体的抗生素抗性和可能的光合自养生长的损失。随后将深入表征光合特性,包括测量稳态氧释放速率、电子传递能力、PSII多肽组成、锰和钙含量以及氯化物需求。 光系统II(PSII)复合体是高等植物、藻类和蓝藻光合机构的一部分。这种复合物催化光驱动的水氧化和质体醌的还原。氧气作为PSII反应的副产品释放出来。CP 43是PSII的蛋白质组分之一,并且似乎是PSII复合物稳定组装所必需的。它似乎也在光合放氧和光能的收集中发挥作用。这种膜结合蛋白的不寻常之处在于它含有一个大的亲水环,该环从它嵌入的膜中突出。大的外部环中氨基酸的短片段的缺失导致不能释放氧的突变体。 该项目的目标是确定参与CP 43的大的、内腔定向的外部环的功能的特定氨基酸,以进一步测试该区域对于正常PSII活性是必要的这一假设。这将通过1)使用随机和位点特异性诱变技术在体外将突变引入编码CP 43的基因中,2)将这些突变基因与蓝绿藻集胞藻6803中的正常对应物交换,和3)评估所得突变生物体组装正常PSII复合物、释放氧和进行电子传递的能力来实现。 *** ??
项目成果
期刊论文数量(0)
专著数量(0)
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会议论文数量(0)
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Cindy Putnam-Evans其他文献
Cindy Putnam-Evans的其他文献
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{{ truncateString('Cindy Putnam-Evans', 18)}}的其他基金
RUI: Role of CP43 in Protein/Protein & Protein/Cofactor Interactions in Photosystem II
RUI:CP43 在蛋白质/蛋白质中的作用
- 批准号:
0517166 - 财政年份:2005
- 资助金额:
$ 18万 - 项目类别:
Continuing Grant
RUI: Interaction of Domains on CP 43 With Components Required for Oxygen Evolution
RUI:CP 43 上的域与析氧所需成分的相互作用
- 批准号:
9982981 - 财政年份:2000
- 资助金额:
$ 18万 - 项目类别:
Standard Grant
The Role of N-terminal Phosphorylation of the Photosystem IIProteins CP-43 and D2 as Probed by Site-Directed Mutagenesis
定点诱变探测光系统 II 蛋白 CP-43 和 D2 N 端磷酸化的作用
- 批准号:
9407187 - 财政年份:1994
- 资助金额:
$ 18万 - 项目类别:
Standard Grant
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