Assembly and Function of the Cyanobacterial Photosystem II Complex

蓝藻光系统 II 复合体的组装和功能

• 批准号: 0818371
• 负责人: Robert Burnap
• 金额: $ 59.32万
• 依托单位: Oklahoma State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0818371&HistoricalAwards=false
• 关键词: Assembly; Function; Cyanobacterial; Photosystem; II

Intellectual Merit: Oxygenic photosynthesis is the major solar energy conversion process in the biosphere consequently it is at the very foundation of life on planet Earth. Photosystem II (PSII) may be considered to be the key enzyme of this process since it uses light energy to split water (water oxidation) liberating the tightly bound hydrogens from the oxygen of water and, at the same time, renders these hydrogens in a form used for producing energy-rich organic carbon compounds from inorganic carbon (carbon dioxide). This project investigates the water-oxidation enzyme of PSII, the replacement of damaged PSII proteins, and assembly of Mn and Ca into the active site.PSII is a dynamic structure that is under a constant state of repair owing to the incessant photodamage it incurs during its normal operation. The repair occurs via a complex assembly pathway involving the removal of damaged D1 protein, insertion and proteolytic processing of the nascent D1 protein precursor and the assembly of the metal atoms of the manganese cluster (4Mn-Ca), which is the heart of the water oxidation reaction. The D1 protein is the locus of most of the above mentioned photodamage. A dedicated and conserved D1 damage detection and replacement mechanism has evolved to repair the damage, but remains to be understood. 1. Mechanism of 4Mn-Ca assembly. The active site of water-oxidation contains a cluster of four manganese and one calcium ions (4Mn-Ca) most of which are bound by the labile D1 protein. The assembly of the 4Mn-Ca requires light to drive the assembly into an active metal cluster. For reasons that remain unclear, this assembly process occurs with very low quantum efficiency. Previous NSF-supported analysis revealed new kinetic features of this process. Hypotheses regarding the origin of these new kinetic features will be evaluated using site-directed mutagenesis together with sensitive biochemical and biophysical techniques that follow the assembly process. The metalloprotein assembly process is being considered in a kinetic framework, that if valid, would represent a new, stochastic concept of metal cluster assembly.2. Detection of structural changes during the removal and replacement of damaged D1 protein during the PSII repair cycle. The D1 protein is buried deeply within the large PSII complex and, consequently, major rearrangements of the PSII complex must occur during the replacement of damaged D1 and the insertion and activation of a new copy of the protein. How is internal damage within the PSII complex detected and what are the signals to initiate repair? What is happening to the D1 protein during replacement? What are the structural changes associated with the assembly of the metal cluster? Two complementary approaches to experimentally address these questions are used: targeted structure-reactivity approaches using mutant strains containing non-native cysteines that will be reacted with thiol-reactive reagents to quantitatively probe site-specific changes in surface exposure. This directed approach will be complemented using powerful mass spectroscopic methods which have the potential to discover assembly associated changes in exposure at other locations in the protein.Broader Impacts: Educational impacts include student exposure to a multidisciplinary attack on an interesting biological problem and training to utilize, operate, and understand advanced mass spectrometers. An education collaboration with the Native Americans in Biological Sciences (NABS) program is planned. The project incorporates a teaching module tentatively entitled: "Light and Life". It will contain exercises and materials for the "Biological Dilemma" (a multi-faceted real-world issue) and for secondary school teachers to utilize in their home science classes. These will borrow materials from the research project and be modeled on the most successful activities of earlier programs, which involved close collaboration with the high school teachers visiting the campus during the summer academies.

智力优点：氧光合作用是生物圈中的主要太阳能转化过程，因此它是地球生命的基础。光系统II（PSII）可以被认为是此过程的关键酶，因为它使用轻能将水（水氧化）从水的氧气中解放出紧密结合的氢，同时，这些氢以一种用于生产富含富含有机碳的有机有机碳化合物（碳二氧化物）的形式。该项目研究了PSII的水氧化酶，替换受损的PSII蛋白，并将Mn和Ca组装到活性位点中。PSII是一种动态结构，由于不断的光dododagmage在正常运行期间爆发，因此在恒定的修复状态下，它处于恒定的修复状态。修复是通过复杂的组装途径进行的，该途径涉及去除受损的D1蛋白，插入和蛋白水解处理的新生D1蛋白前体以及锰簇（4MN-CA）的金属原子的组装，这是水氧化反应的心脏。 D1蛋白是上述大多数发生的光损伤的基因座。专用和保守的D1损伤检测和替换机制已演变为修复损害，但仍有待理解。 1。4MN-CA组装的机制。 水氧化的活性位点含有四个锰和一个钙离子（4MN-Ca）的簇，其中大多数受不稳定的D1蛋白绑定。 4MN-CA的组装需要光将组件驱动到活跃的金属簇中。 由于尚不清楚的原因，该组装过程以非常低的量子效率而发生。 以前的NSF支持分析揭示了此过程的新动力学特征。 关于这些新动力学特征的起源的假设将使用位置定向的诱变以及遵循组装过程的敏感生化和生物物理技术进行评估。 金属蛋白组装过程正在动力学框架中考虑，如果有效，则代表了金属簇组装的新的随机概念。2。在PSII修复周期中去除和更换受损的D1蛋白时的结构变化。 D1蛋白深深地埋在大的PSII复合物中，因此，在更换受损的D1时必须发生PSII复合物的主要重排，并插入和激活该蛋白质的新副本。 PSII复合物内的内部损害如何检测到启动修复的信号是什么？ 替代过程中D1蛋白发生了什么？ 与金属簇的组装相关的结构变化是什么？使用了两种互补的方法来解决这些问题：使用含有非母性半胱氨酸的突变菌株的靶向结构反应性方法，这些方法将与硫醇反应性试剂反应，以定量探测表面暴露的位点特异性变化。 这种定向方法将使用强大的质谱方法进行补充，这些方法有可能发现蛋白质中其他位置的组装相关的暴露变化。BRODER的影响：教育影响包括学生对有趣的生物学问题的多学科攻击以及以利用，操作，操作，操作和理解高级质谱的多学科攻击。 计划与美洲原住民的生物科学（NABS）计划进行教育合作。 该项目结合了一个主题为“光与生命”的教学模块。 它将包含“生物困境”（一个多方面的现实世界问题）的练习和材料，以及中学教师在家庭科学课程中使用的练习和材料。 这些将从研究项目中借用材料，并以早期计划的最成功活动进行建模，该活动涉及与夏季学院期间访问校园的高中教师密切合作。