Directed Mutagenesis of a Photosystem II Extrinsic Protein

小行星9727777 光系统II（PSII）将水氧化成O2的能力需要四核Mn簇的氧化还原活性，所述四核Mn簇由称为锰稳定蛋白（MSP）的26.5kDa外源蛋白的存在调节。 该蛋白质的结构和功能将使用细菌过表达以产生定点突变体来探测。 现有的突变体的表征表明，二硫桥是不需要的活动，影响MSP的N-和C-末端的突变改变其组装与PSII的能力，以及温度敏感性突变，改变二级结构，可以诱导重新绑定到PSII。 基于这些结果，MSP的新实验将集中在：1。 N-和C-末端突变对PSII结合和活性重建的影响; 2. 阐明了电荷反转突变R151 D不能恢复PSII高周转率的机制; 3. 创建新的突变，努力提供新的细节结合到PSII对MSP的结构和活性的影响，以确定工程化的Cys突变是否可以提供新的网站的MSP的共价修饰，并试图确定MSP结构域是敏感的Mn氧化态。 光合作用产生大气氧气的能力需要锰稳定蛋白。 该项目的目标是了解蛋白质如何促进氧气的释放。 蛋白质中的氨基酸将使用分子生物学技术改变。 这些变化如何影响氧气的产生，然后将通过检查突变的蛋白质来分析，以确定它们的结构是否已经改变，并通过评估它们恢复氧气释放的能力。 通过比较来自该蛋白质的许多修饰版本的数据，可以构建将锰稳定蛋白质的性质与氧释放反应的性质相关联的模型。

9727777 Yocum The ability of photosystem II (PSII) to oxidize water to O2 requires the redox activity of a tetranuclear Mn cluster that is regulated by the presence of a 26.5 kDa extrinsic protein called Manganese Stabilizing Protein, or MSP. The structure and function of this protein will be probed using bacterial overexpression to produce site-directed mutants. Characterization of existing mutants shows that a disulfide bridge is not required for activity, that mutations affecting the N- and C-termini of MSP alter its ability to assemble with PSII, and that temperature-sensitive mutations that alter secondary structure can be induced to rebind to PSII. Building on these results, new experiments on MSP will focus on: 1. Effect of N- and C- terminal mutations on PSII binding and activity reconstitution; 2. Elucidating the mechanism by which a charge reversal mutation, R151D fails to restore high turnover rates in PSII; 3. Creating new mutations in efforts to provide new details about the effect of binding to PSII on MSP structure and activity, to determine whether engineered Cys mutations can provide new sites for covalent modification of MSP, and to attempt to identify MSP domains that are sensitive to Mn oxidation states. The ability of photosynthesis to produce atmospheric oxygen requires Manganese Stabilizing Protein. This project's goal is to understand how the protein facilitates oxygen evolution. Amino acids in the protein will be changed using molecular biological techniques. How these changes affect oxygen production will then be analyzed by examining mutated proteins to determine if their structures have been altered, and by assessing their ability to restore oxygen evolution. By comparing data from a number of modified versions of this protein, a model can be constructed that relates properties of Manganese Stabilizing Protein to those of the oxygen-evolving reaction.