Directed Mutagenesis of a Photosystem II Extrinsic Protein

9314743 Yocum光系统II是一种复杂的酶系统，可以捕捉光线，并将这些能量用于一系列反应，其中之一是将H2O氧化为O2。一簇疏水的膜蛋白为最低水平的放氧活性所需的无机辅助因子(钙、氯、锰)提供了连接位点。然而，这些辅因子的稳定连接和动力学上有效的H2O氧化最低限度地需要一个被称为锰稳定蛋白或MSP的外源性33 kDa多肽的存在。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为连接催化H2O氧化的锰原子的位置提供稳定性。虽然已经确定了MSP与光系统结合的一个部位，但该蛋白质的作用方式，无论是纯粹的结构上的，还是作为金属配体的可能贡献者，仍然没有确定。MSP在光系统II中的功能问题将通过定点突变来解决。突变的MSP在大肠杆菌中的表达将被用来获得足够数量的蛋白质，用于重组、结构和光谱研究，该系统使用从拟南芥和菠菜中纯化的光系统II制剂。突变目标残基的选择是基于它们对蛋白质三级结构或MSP与光系统II结合的可能贡献。对重组制剂的分析旨在揭示无机离子辅助因子结合的修饰，催化H2O氧化的锰簇的结构变化，以及用诱变的MSP重组的制剂与其他外源蛋白结合能力的缺陷，该区域也是生理条件下长期持续活性所必需的。这些实验将更好地理解一种重要的光合作用蛋白质的结构，以及它对光合作用放氧部位的组织和催化功能的贡献。光合作用产生维持地球上生命的氧气。执行这一过程的酶被称为光系统II，存在于所有植物和藻类中。虽然已经知道某些离子(锰、钙和氯)是氧气产生所必需的，但光系统II蛋白在氧气产生中的作用还不是很清楚。这个项目将研究一种名为锰稳定蛋白的光系统II蛋白质，它对氧气产生是必不可少的，如果它被去除，氧气产生就会受到损害，暴露在阳光下就会开始破坏酶。为了研究锰稳定蛋白如何保护光系统II和提高氧气的产生，蛋白质的遗传密码首先被转移到细菌E.Coli，它可以被诱导产生天然蛋白质或包含大量突变的替代版本。一旦被分离，这些蛋白质将被检查其结构是否发生突变引起的变化，当它们被添加回光系统II时，将检查它们影响氧气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。通过系统的修饰，将有可能确定蛋白质的哪些部分对稳定光系统II起重要作用，以及蛋白质的哪些部分参与促进产生氧的化学反应。虽然这些实验旨在了解一个非常基本的生物反应，但它们也将提供新的信息，这些信息可能在两个领域被证明是有用的：第一，当充分了解拟南芥和菠菜等模式植物系统产生氧气的机制时，这一知识可能为执行目前非常耗能的氧化/还原反应的新的化学方法指明方向。其次，确定高效产氧所需的蛋白质特征可以提供有关方法的信息，这些方法可能被证明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. ***