RUI: In Vivo and in Vitro Analysis of Modified Chloroplast Rieske Iron-Sulfur Proteins of Chlamydomonas

RUI：衣藻修饰叶绿体 Rieske 铁硫蛋白的体内和体外分析

• 批准号: 0091415
• 负责人: Toivo Kallas
• 金额: $ 33万
• 依托单位: University of Wisconsin-Oshkosh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0091415&HistoricalAwards=false
• 关键词: RUI; Vivo; Vitro; Analysis; Modified

The Rieske iron-sulfur protein of the chloroplast cytochrome b6 f complex has essential roles in energy transduction, redox-sensing, and in biochemical signaling to regulate photosynthesis. A unique movement of the iron-sulfur cluster domain is necessary for catalytic activity and probably for signaling. The research will investigate the role of protein structural features in signal transduction, catalytic activity, and assembly. Modified Rieske proteins will be studied in the alga Chlamydomonas, in isolated complexes, and as overproduced proteins in vitro. Specific objectives are: 1) Mutational investigations in vivo and in vitro of the role of the Rieske protein N-terminus and inflexible hinge domains in signal transduction and catalysis. Included are cysteine insertions to attach fluorescent probes to isolated complexes for assessment of possibleRieske N-terminal movement in signaling. 2) Mutational studies in vivo of an N-terminal Lys-Arg motif and its possible role in .pH-dependent, Rieske protein translocation across the thylakoid membrane. 3) Analysis of overproduced, modified Rieske proteins (such as those lacking the disulfide bridge) by spectroscopic methods to characterize structure/function relationships. Proteins of particular interest may be purified for structure determination. 4) In vitro reconstitution of overproduced Rieske proteins with Rieske-depleted cytochrome b6 f complex. Success would allow in vitro studies of Rieske protein interactions with neighboring subunits and possible LHCII kinase activation in signaling. The research is multifaceted, relies on stimulating collaborations, and will provide important new insights into fundamental mechanisms of photosynthesis and membrane proteins. It will contribute to complementary research and teaching activities at UW-Oshkosh and will provide excellent training opportunities for MS and undergraduate students.

叶绿体细胞色素b6f复合体中的Rieske铁硫蛋白在能量转导、氧化还原感应和调节光合作用的生化信号传导中发挥重要作用。铁硫簇结构域的独特运动是催化活性和信号传递所必需的。本研究将探讨蛋白质结构特征在信号转导、催化活性和组装中的作用。修饰的Rieske蛋白将在衣藻、分离的复合物和体外过量生产的蛋白质中进行研究。具体目标是：1)体内和体外突变研究Rieske蛋白n端和不灵活铰链结构域在信号转导和催化中的作用。包括半胱氨酸插入，将荧光探针连接到分离的复合物上，以评估信号传导中可能的erieske n端运动。2)一个n端Lys-Arg基序的体内突变研究及其可能的作用。ph依赖性，Rieske蛋白在类囊体膜上的易位。3)利用光谱学方法分析过量生产、修饰的Rieske蛋白（如缺乏二硫桥的Rieske蛋白），以表征结构/功能关系。特别感兴趣的蛋白质可以被纯化以用于结构测定。4)用Rieske-耗尽细胞色素b6f复合物体外重建过量生产的Rieske蛋白。成功将允许体外研究Rieske蛋白与邻近亚基的相互作用以及可能的LHCII激酶在信号传导中的激活。这项研究是多方面的，依赖于刺激的合作，并将为光合作用和膜蛋白的基本机制提供重要的新见解。它将有助于威斯康星大学奥什科什分校的互补研究和教学活动，并将为硕士和本科生提供良好的培训机会。