RUI: Role of CP43 in Protein/Protein & Protein/Cofactor Interactions in Photosystem II

本研究旨在探讨高等植物、绿藻和蓝藻光系统II复合体中的叶绿素结合蛋白CP 43的结构和功能。光系统II利用来自太阳的光能驱动水分子的分裂，将分子氧释放到大气中。大气中存在的几乎所有氧气都来自光系统II对水的氧化。CP 43蛋白嵌入类囊体膜中，但它包含几个外环区域，包括大的外环E，它从膜突出到类囊体内(腔)，位于靠近水氧化的位置。诱变技术表明，CP43管腔表面的结构域似乎与放氧复合体的成分相互作用，特别是细胞色素C550和无机辅助因子钙、氯和锰。为了确定这些结构域在光系统II功能中的作用，该项目将对这些突变体进行扩展表征，并在这些位点构建和表征更多的突变。将结合生理、生化和生物物理技术对突变体进行分析。分析突变体的生化方法将包括测量在耗尽Ca~(2+)或Cl-的介质中的生长速度，测量光合作用电子传递速率，测定放氧的相对量子产率，测定光失活速率，评估细胞色素C550与分离的光系统II的结合，蛋白质印迹和差异光谱评估光系统II的蛋白质组成。生物物理方法将侧重于分析锰簇的功能性质，并将包括EPR和FT-IR光谱以及可变荧光产量的测量。由于光系统II还没有得到非常高分辨率的晶体结构，这些研究将提供关于外源蛋白质和内在蛋白质之间相互作用的确切位置的重要信息，以及放氧所需的辅因子。更广泛的影响：预计许多本科生和几名硕士研究生将进行与该项目相关的研究。这些学生不仅将在实验室获得研究经验，他们还将参加科学会议并在科学会议上展示他们的工作，并以作者的身份出现在手稿上。将启动与爱丽丝·哈迪博士(北卡罗来纳大学格林斯伯勒分校)的非正式“交流”计划，她是光系统II的EPR光谱专家。来自ECU的学生将参观哈迪博士的实验室，以学习EPR技术；而她的学生将在ECU学习组氨酸标记光系统II的生化分离。这将使两所大学的学生受益匪浅，因为这将使我们能够拓宽他们的研究经验。

This study is designed to probe the structure and function of the chlorophyll-binding protein CP 43, which is a component of the photosystem II complex of higher plants, green algae and cyanobacteria. Photosystem II uses light energy from the sun to drive the splitting of water molecules with release of molecular oxygen to the atmosphere. Virtually all of the oxygen present in our atmosphere arises from water oxidation by photosystem II. The CP 43 protein is embedded in the thylakoid membrane; however, it contains several extrinsic loop regions, including the large extrinsic loop E, which protrude from the membrane into the interior (lumen) of the thylakoid and lie close to the site of water oxidation. Mutagenesis techniques have shown that domains on the lumenal surface of CP43 appear to interact with components of the oxygen-evolving complex, in particular, cytochrome c550 and the inorganic cofactors Ca2+, Cl- and Mn2+. This project will undertake the expanded characterization of these mutants and the construction and characterization of additional mutations at these sites in order to define the roles of these domains in photosystem II function. A combination of physiological, biochemical and biophysical techniques will be employed to analyze the mutants. Biochemical methods for analyzing mutants will include measurement of growth rates in media depleted in either Ca2+ or Cl-, measurement of photosynthetic electron transport rates, determination of relative quantum yields for oxygen evolution, determination of photoinactivation rates, assessment of the binding of cytochrome c550 to isolated photosystem II, western blotting and difference spectra to assess photosystem II protein composition. Biophysical methods will focus on analyzing the functional properties of the manganese cluster and will include EPR and FT-IR spectroscopy and measurement of variable fluorescence yields. Since a very highly resolved crystal structure has not yet been obtained for photosystem II, these studies will provide important information on the exact sites of interaction between the extrinsic proteins and the intrinsic proteins and cofactors required for oxygen evolution. Broader Impacts: It is anticipated that numerous undergraduate and several M.S. students will perform research related to the project. These students will not only gain research experience in the laboratory, they will also attend and present their work at scientific meetings and appear as authors on manuscripts. An informal "exchange" program with Dr. Alice Haddy (UNC-Greensboro), an expert in EPR spectroscopy of photosystem II, will be initiated. Students from ECU will visit Dr. Haddy's laboratory in order to learn EPR techniques; while her students will learn the biochemical isolation of histidine-tagged photosystem II at ECU. This will greatly benefit students from both institutions, as it will allow us to broaden their research experience.