RUI: Interaction of Domains on CP 43 With Components Required for Oxygen Evolution

本项目旨在利用诱变技术研究叶绿素结合蛋白cp43的结构和功能。CP 43是高等植物、绿藻和蓝藻的光系统II （PSII）复合体的一种蛋白质成分。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。实际上，我们大气中存在的所有氧气都是由PSII氧化水产生的。cp43蛋白包埋在类囊体膜中；然而，它包含几个外部环区，包括一个大的外部环E，它从膜突出到类囊体的内部（管腔）（水氧化的部位）。众所周知，CP 43在光收集过程中发挥作用。然而，越来越多的数据表明，CP 43在PS II络合物的稳定组装和水裂解（氧气演化）过程中发挥着额外的作用。PI假设cp43管腔表面的结构域，特别是大的外部环E，与氧进化复合物的组分相互作用。PI的实验室正在通过使用基因工程技术在所有腔内外部环中产生突变来验证这一假设。这是通过在编码cp43管腔环的psbC基因区域引入特定的碱基对变化来实现的，然后将改变的基因引入我们的模式生物蓝藻-聚囊藻6803。然后，该生物体产生一种突变蛋白，在其中一个外源性环中含有单个氨基酸替代。迄今为止，PI主要关注管腔结构域中带电氨基酸残基的改变。在胞囊藻（Synechocystis 6803） cp43蛋白外源性大环E的22个位点上构建了21个突变体。其中四个突变体聚集了PSII中心，但PSII活性受损。另外一个突变体不能组装任何功能中心，因此缺乏PSII活性。管腔环中的其他11种氨基酸是诱变的目标。生理、生化和生物物理技术的结合将被用来分析和进一步表征这些突变体。

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.