GENETICS AND BIOCHEMISTRY OF A MEMBRANE PROTEIN COMPLEX

膜蛋白复合物的遗传学和生物化学

• 批准号: 2392140
• 负责人: HIMADRI B PAKRASI
• 金额: $ 15.48万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392140
• 关键词: bacterial genetics; cytochrome b; environmental stressor; gene mutation; genetic strain; laboratory rabbit; light intensity; manganese; membrane proteins; molecular cloning; photobiology; photostimulus; photosynthetic bacteria; photosynthetic reaction centers; photosystem; protein degradation; protein structure function; proteolysis; radiation sensitivity; recombinant DNA; regulatory gene; site directed mutagenesis; suppressor mutations; transposon /insertion element

This proposal is focused on the study of the membrane protein complex Photosystem II (PSII). PSII is an intriguing light-driven molecular machine. Localized in the thylakoid membranes, this pigment-protein complex mediates electron transfer from water to quinones. Even though light is a substrate for PSH, it creates damage that results in the degradation of this protein complex. To survive, photosynthetic organisms have developed elaborate repair systems. The long range goal of this project is to understand the biochemical mechanisms that protect PII from such light-induced damages. Our experimental organism is Synechocystis 6803, a unicellular, naturally transformable cyanobacterium. This proposal contains plans for detailed genetic and biochemical studies of the functional roles of D1 and cytochrome b559, two core polypeptide components of PSII, as well as CtpA, a recently identified carboxyl- terminal processing protease, in the damage-repair cycle of the PSII complex exposed to light. Our aims are : (1) To elucidate the function of the carboxyl-terminal extension of the D1 protein. Following its synthesis and integration into the PSII complex, the precursor form of D1 is processed, after which PSII becomes catalytically active. We will investigate how the presence of this extension peptide in D1 influences the biogenesis and function of PSII. (2) To dissect the mechanism of CtpA, a novel processing protease that cleaves the C-terminal extension in D1. (3) To use light-tolerant mutants to identify key determinants in the protection of PSII from photodamage. We will generate random mutations in the psbEF gene cluster encoding cyt b559, to isolate Synechocystis mutants that can grow under high light intensities. Characterization of these mutant strains will aid in our understanding of the roles of these two proteins in alleviating light stress. (4) To elucidate the function of cyt b559 in PSII. Our experiments will be aimed at understanding the mechanism of photoprotection mediated by this protein. We will also use second site suppressor analysis to determine the overall function of cyt b559 in PSII. In view of the rapid turnover of individual polypeptide components of PSII constantly facing potential damage by visible light, the current challenge is to understand the mechanisms that carefully control the formation, stability and function of this pigment-protein complex. We will use tools of genetics, biochemistry and spectroscopy to pursue this challenge. These studies will also provide valuable insights about proteolytic degradation and subsequent repair of membrane bound proteins, key processes related to a number of human diseases as well as aging.

该提案的重点是膜蛋白复合物的研究 光系统 II (PSII)。 PSII 是一种有趣的光驱动分子 机器。这种色素蛋白位于类囊体膜中 复合物介导从水到醌的电子转移。虽然 光是 PSH 的底物，它会造成损害，从而导致 该蛋白质复合物的降解。 为了生存，光合生物 开发了复杂的修复系统。本次活动的长远目标 该项目旨在了解保护 PII 免受侵害的生化机制 此类光引起的损害。 我们的实验生物是集胞藻 6803，一种单细胞、可自然转化的蓝细菌。这个提议 包含详细的遗传和生化研究计划 两个核心多肽 D1 和细胞色素 b559 的功能作用 PSII 的成分，以及 CtpA（最近发现的一种羧基） PSII 损伤修复周期中的末端加工蛋白酶 复合体暴露于光下。我们的目标是： (1) 阐明 D1 蛋白的羧基末端延伸。其合成后 并整合到 PSII 复合物中，D1 的前体形式是 处理后，PSII 变得具有催化活性。我们将 研究 D1 中这种延伸肽的存在如何影响 PSII 的生物发生和功能。 (2) 剖析CtpA的作用机制， 一种新型加工蛋白酶，可切割 D1 中的 C 端延伸。 (3) 利用耐光突变体来鉴定关键决定因素 保护 PSII 免受光损伤。我们将生成随机突变 编码 cyt b559 的 psbEF 基因簇，用于分离集胞藻突变体 可以在高光照强度下生长。这些的表征 突变株将有助于我们理解这两种病毒的作用 蛋白质可以缓解光应激。 (4)阐明cyt的功能 PSII 中的 b559。我们的实验旨在了解其机制 由该蛋白质介导的光保护作用。我们还将使用第二个站点 抑制子分析以确定 PSII 中 cyt b559 的整体功能。 鉴于 PSII 单个多肽成分的快速周转 不断面临可见光的潜在损害，这是当前的挑战 是了解仔细控制形成的机制， 这种色素-蛋白质复合物的稳定性和功能。 我们将使用工具 遗传学、生物化学和光谱学的研究来应对这一挑战。 这些研究还将提供有关蛋白水解的宝贵见解 膜结合蛋白的降解和随后的修复，关键 与许多人类疾病以及衰老相关的过程。