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
• 负责人: Cindy Putnam-Evans
• 金额: $ 27.92万
• 依托单位: East Carolina University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-15 至 2009-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517166&HistoricalAwards=false
• 关键词: RUI; Role; CP43; Protein; &amp

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.

本研究旨在探讨叶绿素结合蛋白CP 43的结构和功能，CP 43是高等植物、绿藻和蓝藻光系统II复合体的组成部分。光系统II利用来自太阳的光能驱动水分子的分裂，并向大气释放分子氧。实际上，我们大气中存在的所有氧气都是由光系统II对水的氧化作用产生的。cp43蛋白包埋在类囊体膜中；然而，它包含几个外部环区，包括大的外部环E，它从膜伸出到类囊体的内部（管腔），靠近水氧化的部位。诱变技术表明，CP43管腔表面的结构域似乎与氧进化复合物的组分相互作用，特别是细胞色素c550和无机辅因子Ca2+， Cl-和Mn2+。该项目将对这些突变体进行扩展表征，并在这些位点上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。生理、生化和生物物理技术的结合将被用来分析突变体。分析突变体的生化方法将包括测量在Ca2+或Cl-培养基中的生长速率，测量光合电子传递速率，测定氧演化的相对量子产率，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，western blotting和差异光谱来评估光系统II的蛋白质组成。生物物理方法将侧重于分析锰簇的功能特性，包括EPR和FT-IR光谱以及可变荧光产率的测量。由于光系统II尚未获得非常高分辨率的晶体结构，因此这些研究将提供关于氧演化所需的外在蛋白质和内在蛋白质以及辅因子之间相互作用的确切位置的重要信息。更广泛的影响：预计许多本科生和一些硕士学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展示他们的工作，并以手稿作者的身份出现。将启动与光系统II EPR光谱专家Alice Haddy博士（UNC-Greensboro）的非正式“交流”计划。ECU的学生将参观Haddy博士的实验室，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。这将大大有利于两所院校的学生，因为这将使我们扩大他们的研究经验。