GENETIC ANALYSIS OF THE PHOTOSYNTHETIC REACTION CENTER

光合反应中心的遗传分析

• 批准号: 3308021
• 负责人: EDWARD J BYLINA
• 金额: $ 9.85万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3308021
• 关键词: Rhodopseudomonas; X ray crystallography; bacterial DNA; bacterial genetics; chlorophyll; cytochrome c; electron spin resonance spectroscopy; electron transport; genetic strain; molecular cloning; mutant; operon; photosynthesis; photosynthetic reaction centers; photosystem; plasmids; protein engineering; protein sequence; protein structure function; recombinant DNA; reducing agents; site directed mutagenesis; structural biology; transfection; transfection /expression vector; transposon /insertion element

Membrane-bound protein complexes are involved in many important health related processes including respiration, transport processes, transformation, sensory transduction, receptor binding, and signal transduction. the bacterial reaction center complex, which mediates a series of efficient electron transfer reactions resulting in the conversion of light into chemical energy, is the only membrane protein whose structure has been determined to atomic resolution. While the highest resolution structural data (2.3 angstroms) is available from the reaction centers of Rhodopseudomonas viridis, a system for the expression of genetically modified reaction centers in this organism is not available. We have been characterizing a heterotrophic strain of Rps. viridis which will be used to develop such a genetic system. the Rps. viridis reaction center system will provide the opportunity to test substrate recognition/binding requirements in membrane proteins and theories of electron transfer in biological systems, since the protein structure of modified complexes can be determined and then correlated with biochemical and spectroscopic studies of these same complexes. The puf operon of Rps. viridis (encoding the beta and alpha subunits of the light harvesting complex and the L subunit, M subunit, and cytochrome subunit of the reaction center) will be deleted from the Rps. viridis chromosome. A plasmid containing a copy of the puf operon will be used to complement this chromosomal deletion. Pigment-protein binding interactions throughout the reaction center will be modified by site- directed mutagenesis of the Rps. viridis structural genes, followed by crystallographic and spectroscopic characterization of the altered reaction center complexes. The insights provided by the Rps. viridis reaction center system will provide a basis for understanding of structure-function relationships in other membrane proteins where atomic resolution structural information is not available.

膜结合蛋白复合物参与许多重要的健康 相关过程包括呼吸作用、运输过程、 转化、感觉传导、受体结合和信号传导 转导 细菌反应中心复合体，它介导 一系列有效的电子转移反应， 光转化为化学能，是唯一的膜蛋白 其结构已被确定为原子分辨率。 而 最高分辨率的结构数据（2.3埃）可从 反应中心的红球藻，一个系统的表达 基因改造的反应中心， available. 我们一直在描述一种异养型的细菌。 viridis将用于开发这样的遗传系统。 的RPS。 viridis反应中心系统将提供机会， 膜蛋白中的底物识别/结合要求， 生物系统中的电子转移理论，因为蛋白质 可以确定改性络合物的结构，然后关联 对这些复合物进行了生化和光谱研究。 的 Rps的puf操纵子viridis（编码β和α亚基的 捕光复合物和L亚基、M亚基和细胞色素 反应中心的亚基）将从Rps中删除。viridis 染色体 将使用含有puf操纵子拷贝的质粒 来补充染色体缺失 色素蛋白结合 整个反应中心的相互作用将被现场修改- Rps的定向诱变。viridis结构基因，其次是 晶体学和光谱学特征的改变 反应中心络合物 Rps提供的见解。viridis 反应中心系统将为理解 结构-功能关系在其他膜蛋白，其中原子 分辨率结构信息不可用。