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: 2900749
• 负责人: HIMADRI B PAKRASI
• 金额: $ 16.72万
• 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 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900749
• 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.

该建议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 机这种色素蛋白位于类囊体膜上， 络合物介导电子从水转移到醌。即使 光是PSH的基质，它会造成损伤，导致 降解这种蛋白质复合物。 为了生存，光合生物 已经发展出复杂的修复系统。长期目标是 该项目旨在了解保护PII免受 这种光损伤。 我们的实验生物是集胞藻 6803，一种单细胞的自然可转化的蓝细菌。这项建议 载有详细的遗传和生物化学研究计划， D1和细胞色素b559两个核心多肽的功能作用 PSII的成分，以及CtpA，最近确定的羧基- 末端加工蛋白酶，在PSII的损伤修复循环中 复杂暴露于光。我们的目的是：（1）阐明的功能， D1蛋白的羧基末端延伸。在其综合 并整合到PSII复合物中，D1的前体形式是 处理，之后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)为了剖析CtpA的机制， 一种切割D1中C-末端延伸的新型加工蛋白酶。 (3)利用耐光突变体来鉴定在光敏感性中的关键决定因素， 保护PSII免受光损伤。我们将在这些基因中产生随机突变 编码cyt b559的psbEF基因簇，以分离集胞藻突变体 可以在强光下生长表征这些 突变菌株将有助于我们理解这两个 蛋白质减轻光胁迫。(4)阐明细胞色素T的功能 PSII中的b559。我们的实验旨在了解 由这种蛋白质介导的光保护作用。我们还将使用第二个网站 抑制子分析以确定细胞色素b559在PSII中的总体功能。 鉴于PSII的单个多肽组分的快速周转， 不断面临可见光的潜在损害， 就是要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结合来应对这一挑战。 这些研究也将为蛋白水解提供有价值的见解 降解和随后的修复膜结合蛋白，关键 与许多人类疾病以及衰老有关的过程。