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.

智力优势：光合作用是生物圈中主要的太阳能转换过程，因此它是地球上生命的基础。光系统II（PSII）可以被认为是该过程的关键酶，因为它使用光能来分解水（水氧化），从水中的氧中释放出紧密结合的氢，同时，使这些氢以用于从无机碳（二氧化碳）产生富含能量的有机碳化合物的形式存在。本计画研究PSII的水氧化酵素、受损PSII蛋白质的置换、Mn与Ca组装成活性部位。PSII是一种动态结构，在正常运作时，由于不断受到光损伤，处于持续的修复状态。修复通过复杂的组装途径发生，包括去除受损的D1蛋白，新生D1蛋白前体的插入和蛋白水解加工以及锰簇（4 Mn-Ca）的金属原子的组装，这是水氧化反应的核心。 D1蛋白是上述大多数光损伤的位点。一个专门的和保守的D1损伤检测和替换机制已经发展到修复损伤，但仍有待了解。1. 4 Mn-Ca组装机制 水氧化的活性位点包含四个锰离子和一个钙离子的簇（4 Mn-Ca），其中大部分被不稳定的D1蛋白结合。 4 Mn-Ca的组装需要光来驱动组装成活性金属簇。 由于尚不清楚的原因，这种组装过程的量子效率非常低。 以前的NSF支持的分析揭示了这个过程的新的动力学特征。 关于这些新的动力学特征的起源的假设将使用定点诱变连同敏感的生物化学和生物物理技术，遵循的组装过程进行评估。 金属蛋白组装过程被认为是在一个动力学框架，如果有效，将代表一个新的，随机的金属簇组装的概念。检测在PSII修复周期中受损D1蛋白的去除和替换过程中的结构变化。 D1蛋白深埋在大的PSII复合物中，因此，在替换受损的D1和插入并激活新的蛋白质拷贝期间，必须发生PSII复合物的主要重排。PSII复合体内部的损伤是如何检测的？启动修复的信号是什么？ D1蛋白在置换过程中发生了什么？ 与金属簇组装相关的结构变化是什么？两种互补的方法来解决这些问题的实验使用：有针对性的结构反应性的方法，使用含有非天然半胱氨酸，将与巯基反应试剂反应，以定量探针位点特异性变化的表面暴露的突变株。 这种直接的方法将补充使用强大的质谱方法，有可能发现组装相关的变化暴露在其他位置的蛋白质。更广泛的影响：教育的影响包括学生接触到一个有趣的生物学问题和培训的多学科攻击利用，操作和理解先进的质谱仪。 计划与美国原住民生物科学（NABS）计划进行教育合作。 该项目包含一个暂定标题为“光与生命”的教学模块。 它将包含“生物困境”（一个多方面的现实问题）的练习和材料，供中学教师在家庭科学课上使用。 这些将从研究项目中借用材料，并以早期项目中最成功的活动为蓝本，这些活动涉及与夏季学院期间访问校园的高中教师密切合作。