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

本研究旨在探讨高等植物、绿色藻类和蓝藻的光系统II复合体中叶绿素结合蛋白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分裂，并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光系统II对水的氧化。 CP 43蛋白包埋在类囊体膜中;然而，它含有几个外部环区域，包括大的外部环E，其从膜突出到类囊体的内部（内腔）并靠近水氧化的位点。 诱变技术已经表明，CP43内腔表面上的结构域似乎与放氧复合物的组分相互作用，特别是细胞色素c550和无机辅因子Ca 2+、Cl-和Mn 2+。 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额外的突变，以确定这些结构域在光系统II功能中的作用。 将采用生理学、生物化学和生物物理学技术相结合的方法来分析突变体。用于分析突变体的生化方法将包括测量在Ca2+或Cl-耗尽的培养基中的生长速率、测量光合电子传递速率、测定析氧的相对量子产率、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、蛋白质印迹和差谱以评估光系统II蛋白质组成。生物物理方法将侧重于分析锰簇的功能特性，并将包括EPR和FT-IR光谱和测量可变荧光产率。 由于一个非常高分辨率的晶体结构尚未获得光系统II，这些研究将提供重要的信息，外部蛋白质和内在的蛋白质和辅因子之间的相互作用的确切位点的氧气演变。 更广泛的影响：预计许多本科生和几个硕士。学生将进行与项目相关的研究。 这些学生不仅将在实验室获得研究经验，他们还将参加并在科学会议上展示他们的工作，并作为手稿的作者出现。 将启动与光系统II EPR光谱学专家Alice Haddy博士（北卡罗来纳大学格林斯伯勒分校）的非正式"交流"计划。 来自ECU的学生将参观Haddy博士的实验室，以学习EPR技术;而她的学生将在ECU学习组氨酸标记的光系统II的生物化学分离。 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.