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: 2684961
• 负责人: HIMADRI B PAKRASI
• 金额: $ 16.09万
• 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/2684961
• 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.

这项建议的重点是膜蛋白复合体的研究。 光系统II(PSII)。PSII是一种有趣的光驱动分子 机器。这种色素蛋白定位在类囊体膜上， 络合物介导电子从水到对苯二酚的转移。即使 光是PSH的底物，它会造成损害，导致 这种蛋白质复合体的降解。为了生存，光合作用的有机体 已经开发出了复杂的修复系统。这样做的长期目标是 项目是了解保护PII免受感染的生化机制。 这种由光引起的损害。我们的实验生物体是聚球藻 6803，一种单细胞的，自然转化的蓝藻。这项建议 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，一种新近发现的羧基- PSII损伤修复循环中的末端加工蛋白酶 暴露在光下的复合体。我们的目的是：(1)阐明 D1蛋白的羧基末端延伸。在它的合成之后 并整合到PSII复合体中，D1的前体形式是 经过处理后，PSII变得具有催化活性。我们会 研究该延伸肽在D1中的存在如何影响 PSII的生物发生和功能。(2)对CTPA的作用机制进行剖析。 一种新的加工酶，它能裂解D1中的C-末端延伸。 (3)利用耐光突变体来鉴定植物体内的关键决定因素 保护PSII免受光损伤。我们将产生随机突变 编码Cyt b559的psbEF基因簇，用于分离集胞藻突变体 它们可以在高光强下生长。对这些的描述 突变菌株将有助于我们理解这两种基因的作用。 缓解光胁迫的蛋白质。(4)阐明细胞色素T的功能 B559在PSII中。我们的实验将旨在了解这一机制 由这种蛋白质介导的光保护作用。我们还将使用第二个站点 抑制子分析以确定细胞色素b 559在PSII中的整体功能。 鉴于PSII的单个多肽成分的快速周转 不断面临可见光的潜在损害，当前的挑战 就是了解仔细控制形成的机制， 该色素-蛋白质复合体的稳定性和功能。我们将使用工具 遗传学、生物化学和光谱学来应对这一挑战。 这些研究也将为蛋白质分解提供有价值的见解。 膜结合蛋白的降解和后续修复，关键 这一过程与许多人类疾病以及衰老有关。