Directed Mutagenesis of a Photosystem II Extrinsic Protein

Yocum光系统II是一个复杂的酶系统，它收集光并利用这些能量进行一系列反应，其中之一是将H2O氧化成O2。一组疏水膜蛋白为无机辅因子（Ca2+, C1, Mn）提供连接位点，这是最低水平的O2进化活性所必需的。然而，这些辅助因子的稳定连接和动力学高效的H2O氧化最低限度地需要一种称为锰稳定蛋白（MSP）的外源性33 kDa多肽的存在。该蛋白在体内和纯化光系统II制剂中的一个关键功能是赋予连接Mn原子催化H2O氧化的位点稳定性。虽然已经确定了MSP与光系统结合的一个位点，但该蛋白的作用模式，无论是纯粹的结构还是作为金属配体的可能贡献者，仍未确定。MSP在光系统II中的功能问题将通过位点定向诱变来解决。在大肠杆菌中表达诱变的MSP将用于获得足够数量的蛋白质，用于在使用从拟南芥和菠菜中纯化的光系统II制剂的系统中进行重组、结构和光谱研究。靶向诱变的残基是根据它们对蛋白质三级结构或MSP与光系统II结合的可能贡献来选择的。重组制剂的分析旨在揭示无机离子辅助因子结合的改变，催化H2O氧化的锰簇结构的改变，以及用诱变的MSP重组的制剂结合其他外部蛋白质的能力缺陷，这些蛋白质在生理条件下也是长期持续活性所必需的。本实验将更好地了解一种重要的光合蛋白的结构及其在光合O2进化位点的组织和催化功能中的作用。光合作用产生维持地球上生命的氧气。完成这一过程的酶被称为光系统II，存在于所有植物和藻类中。虽然已知某些离子（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尚不清楚。该项目将研究一种光系统II蛋白，称为锰稳定蛋白，它对氧气生产至关重要，如果它被移除，氧气生产就会受损，暴露在光线下开始破坏这种酶。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，首先将蛋白质的遗传密码转移到细菌大肠杆菌中，可以诱导其产生天然蛋白或含有大量突变的替代蛋白。一旦分离出来，这些蛋白质将被检查其结构的突变引起的变化，当它们被添加回光系统II时，检查它们影响氧气生产活性和稳定性的能力的变化。所采用的方法允许研究者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一部分对稳定光系统II很重要，蛋白质的哪一部分参与促进产生氧气的化学反应。虽然这些实验是为了了解一个非常基本的生物反应，但它们也将提供新的信息，可能在两个方面证明是有用的：首先，当模型植物系统如拟南芥和菠菜产生氧气的机制被完全理解时，这些知识可能为进行目前非常耗能的氧化/还原反应指明新的化学方法。其次，确定有效产氧所需的蛋白质特征可以提供有关保护植物生命免受全球变暖等环境因素影响的方法的信息。＊＊＊

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. ***