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
• 负责人: Cindy Putnam-Evans
• 金额: $ 18万
• 依托单位: East Carolina University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2004-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982981&HistoricalAwards=false
• 关键词: RUI; Interaction; Domains; CP; 43

9982981Putnam-EvansThis project is designed to probe the structure and function of the chlorophyll-binding protein CP 43 using mutagenesis techniques. CP 43 is a protein component of the Photosystem II (PSII) complex of higher plants, green algae and cyanobacteria (blue-green algae). PSII uses light energy from the sun to drive the splitting of water molecules with concomitant release of molecular oxygen to the atmosphere. Virtually all of the oxygen present in our atmosphere arises from water oxidation by PSII. The CP 43 protein is embedded in the thylakoid membrane; however, it contains several extrinsic loop regions, including a large extrinsic loop E, which protrude from the membrane into the interior (lumen) of the thylakoid (the site of water oxidation). CP 43 is known to play a role in the light-harvesting process. However, accumulating data point to additional roles for CP 43 in the stable assembly of the PS II complex and in the water splitting (oxygen-evolving) process. The PI hypothesizes that domains on the lumenal surface of CP 43, and in particular the large extrinsic loop E, interact with components of the oxygen-evolving complex. The PI's laboratory is testing this hypothesis by using genetic engineering techniques to produce mutants in all the lumenal extrinsic loops. This is accomplished by introducing specific base pair changes in regions of the psbC gene encoding the lumenal loops of CP 43, and then introducing the altered genes back into our model organism, the cyanobacterium Synechocystis 6803. The organism then produces a mutant protein containing a single amino acid substitution in one of the extrinsic loops. To date the PI has focused on alteration of charged amino acid residues in the lumenal domains. Twenty-one mutants at twenty-two sites in the large extrinsic loop E of the CP 43 protein of Synechocystis 6803 have been constructed. Four of these mutants assemble PSII centers but are impaired in PSII activity. One additional mutant fails to assemble any functional centers and thus is devoid of PSII activity. Eleven other amino acids in lumenal loops are targeted for mutagenesis. A combination of physiological, biochemical and biophysical techniques will be employed to analyze and further characterize these mutants.

9982981Putnam-Evans本项目旨在利用突变技术探索叶绿素结合蛋白CP 43的结构和功能。CP 43是高等植物、绿藻和蓝藻(蓝藻)光系统II(PSII)复合体的蛋白质组分。PSII利用来自太阳的光能驱动水分子的分裂，同时将分子氧释放到大气中。事实上，我们大气中存在的所有氧气都来自PSII对水的氧化。CP 43蛋白嵌入类囊体膜；然而，它包含几个外环区域，包括一个大的外环E，它从膜突出到类囊体膜的内部(腔)(水氧化位置)。已知CP 43在捕光过程中起作用。然而，积累的数据表明，CP 43在PS II复合体的稳定组装和水分解(放氧)过程中发挥了额外的作用。PI假设CP 43管腔表面的结构域，特别是大的外环E，与放氧复合体的成分相互作用。PI的实验室正在通过使用基因工程技术在所有的腔外循环中产生突变来检验这一假设。这是通过在PSBC基因编码CP 43的管腔环的区域引入特定的碱基对变化，然后将改变的基因引入我们的模式生物--蓝藻集胞体6803来实现的。然后，该有机体产生一种突变蛋白，其中一个外在环中含有单一氨基酸替代。到目前为止，PI主要集中在管腔区域带电氨基酸残基的变化上。在聚球藻6803的CP43蛋白的22个外环E处构建了21个突变体。这些突变体中有四个组装了PSII中心，但PSII活性受损。另外一个突变体无法组装任何功能中心，因此缺乏PSII活性。管腔环中的其他11种氨基酸是突变的目标。将结合生理、生化和生物物理技术来分析和进一步鉴定这些突变体。