Directed Mutagenesis of a Photosystem II Extrinsic Protein

Yocum Photosystem II是一种复杂的酶系统，它收集光并将这种能量用于一系列反应，其中之一是 将H2O氧化成O2。 一簇疏水膜蛋白提供连接位点的无机辅因子（Ca 2+，C1，Mn）所需的最低水平的O2进化活动。 然而，这些辅因子的稳定连接和动力学有效的H2O 氧化最低限度地需要存在称为锰稳定蛋白或MSP的外源性33 kDa多肽。 这种蛋白质在体内和纯化的光系统II制剂中的一个关键功能是赋予连接催化H2O氧化的Mn原子的位点稳定性。 虽然 MSP与光系统结合的一个位点已经被确定，该蛋白的作用模式，无论是纯粹的结构性的还是另外作为金属配体的可能贡献者，仍然不确定。 MSP在光系统II中的功能问题将通过定点诱变来解决。 突变MSP在E. 将使用大肠杆菌获得足够量的蛋白质，用于在使用来自拟南芥和菠菜的纯化的光系统II制剂的系统中进行重构、结构和光谱研究。已经根据它们对蛋白质的三级结构或MSP与光系统II的结合的可能贡献来选择用于诱变的靶向残基。 重组制剂的分析旨在揭示无机离子辅因子结合的修饰，催化H2O氧化的锰簇结构的改变，以及用诱变MSP重组的制剂结合生理条件下长期持续活性所必需的其他外源蛋白的能力的缺陷。 这些实验将有助于我们更好地了解一种重要的光合作用蛋白的结构及其对光合放氧位点的组织和催化功能的贡献。 光合作用产生维持地球生命的氧气。 进行这一过程的酶被称为光系统II，在所有植物和藻类中都有发现。虽然，它是已知的，某些离子（锰，钙和氯离子）需要氧气生产发生，光系统II蛋白在氧气生产中的作用还没有得到很好的理解。 该项目将研究一种称为锰稳定蛋白的光系统II蛋白质，该蛋白质对氧气的产生至关重要，如果将其去除，氧气的产生就会受损，并且暴露在光线下会开始破坏酶。 为了研究锰稳定蛋白如何保护光系统II并增强氧气的产生，首先将该蛋白的遗传密码转移到细菌E。大肠杆菌，其可以被诱导产生天然蛋白质或含有大量突变的替代版本。 一旦分离，这些蛋白质将被检查其结构中的突变诱导的变化，并且当它们被添加回光系统II时，检查它们影响氧产生的活性和稳定性的能力的变化。 所采用的方法允许 研究人员在蛋白质中产生许多突变。通过系统修饰，将有可能确定蛋白质的哪些部分在稳定光系统II中是重要的，以及蛋白质的哪些部分参与促进产生氧气的化学反应。 虽然这些实验旨在了解一个非常基本的生物反应，但它们也将提供可能在以下两个领域有用的新信息：首先，当模式植物系统如拟南芥和菠菜产生氧气的机制被完全理解时，该知识可以为进行目前非常耗能的氧化/还原反应的新化学方法指明方向。 其次，确定有效产氧所需的蛋白质特征可以提供有关方法的信息，这些方法可能有助于保护植物免受全球变暖等环境因素的影响。 ***

9314743 Yocum Photosystem II is a complex enzyme system that harvests light and uses this energy for a series of reactions, one of which is the oxidation of H2O to O2. A cluster of hydrophobic membrane proteins provide ligation sites for the inorganic cofactors (Ca2+, C1, Mn) required for minimal levels of O2 evolution activity. However, stable ligation of these cofactors and kinetically efficient H2O oxidation minimally requires the presence of an extrinsic 33 kDa polypeptide known as the Manganese Stabilizing Protein, or MSP. A critical function of this protein in vivo and in purified photosystem II preparations is to confer stability to the site that ligates the Mn atoms that catalyze oxidation of H2O. Although one site of binding to photosystem has been identified for MSP, the protein's mode of action, whether purely structural or in addition as a possible contributor of metal ligands, remaining unsettled. The question of MSP function in photosystem II will be addressed by site-directed mutagenesis. Expression of mutagenized MSP in E. coli will be used to obtain amounts of protein sufficient for reconstitution, structural and spectroscopic investigations in a system using purified photosystem II preparations from Arabidopsis and spinach. Residues targeted for mutagenesis have been selected on the basis of their possible contributions to the tertiary structure of the protein or to binding of MSP to photosystem II. Analyses of reconstituted preparations are designed so as to reveal modifications in inorganic ion cofactor binding, alterations in the structure of the manganese cluster that catalyzes H2O oxidation, and defects in the ability of preparations reconstituted with mutagenized MSP to bind other extrinsic proteins that area also essential for long-term sustained activity under physiological conditions. The proposed experiments will provide a better understanding of the structure of an important photosynthetic protein and its contributions t o both the organization and catalytic function of the site of photosynthetic O2 evolution. %%% Photosynthesis produces the oxygen that sustains life on earth. The enzyme that carries out this process is called photosystem II, and is found in all plants and algae. Although, it is known that certain ions (manganese, calcium and chloride) are needed for oxygen production to occur, the role of photosystem II proteins in oxygen production is not well understood. This project will examine a photosystem II protein, called the Manganese Stabilizing Protein, that is essential to oxygen production, if it is removed, oxygen production is impaired and exposure to light begins to destroy the enzyme. To examine how Manganese Stabilizing Protein protects photosystem II and enhances oxygen production, the genetic code for the protein is first transferred to a bacterium, E. coli, which can be induced to produce the native protein or alternative versions containing mutations in large amounts. Once isolated, these protein will be examines for mutation-induced changes in their structure and, when they are added back to photosystem II, for changes in their ability to affect the activity and stability of oxygen production. The method to be employed permits the investigator to produce many mutations in the protein. By systematic modifications, it will be will be possible to determine which parts of the protein are important in stabilizing photosystem II, and which parts of the protein are involved in promoting the chemical reactions that produce oxygen. Although these experiments are directed at understanding a very fundamental biological reaction, they also will provide new information that may prove useful in 2 areas: first, when the mechanism by which model plant system such as Arabidopsis and spinach produce oxygen is fully understood, this knowledge may point the way to new chemical methods for carrying out oxidation/reduction reactions that are at present very energy-inte nsive. Second, determination of protein features that are required for efficient oxygen production can provide information on methods that may prove useful in protecting plant life from environmental factors such as global warming. ***