Protein-Protein Interactions in Photosystem II

本计画将阐明高等植物中与光系统II（PS II）相关的蛋白质的结构组织。将使用生物化学和分子方法研究光系统结构组分之间的蛋白质-蛋白质相互作用。首先，将检查锰稳定蛋白与24 kDa蛋白的相互作用。锰稳定蛋白质的化学修饰，当它与PS II膜显着影响的24 kDa的蛋白质结合PS II的能力。将确定24 kDa蛋白的结合抑制是否是由于电荷中和或空间干扰。将通过质谱法绘制修饰残基的位置。其次，将确定化学修饰对24和17 kDa蛋白质的蛋白质结合和功能的影响，并绘制修饰残基的位置。第三，所观察到的异质性PS II的外部亚基的光系统的结构和功能方面的作用将被检查。在拟南芥中，PS II的每个外源蛋白由两个基因编码。T-DNA插入线，这是可用于这些基因的数量将被检查。对于没有可用的T-DNA插入系的基因（psbO-2和psbP-1），将引入RNAi构建体以抑制这些基因产物的表达。这些植物的表型将检查PS II功能，组装和控制和胁迫条件下的稳定性。除了这种遗传方法，生化重建研究检查两个psbO蛋白的功能将进行。这些研究将提供重要的新信息，关于PS II的外在成分与光系统的内在成分的相互作用，并与对方。此外，外源亚基异质性的作用的理解可能会提供监管的“微调”的光合作用的见解。最后，我们将产生的拟南芥突变体将为研究外源亚基在体内的作用提供一个资源。更广泛的影响：这个项目将涉及不同层次的培训。预计将有几名本科生参与与该项目有关的研究。这些学生将参加科学会议，介绍他们的研究，并最终发表他们的结果。研究生，博士后研究人员和研究助理都密切参与这些本科研究人员的培训。此外，该项目将继续与东卡罗莱纳大学的Cindy Putnam-Evans博士长期合作，她是一名非博士的女教师，授予部门。

This project will elucidate the structural organization of proteins associated with Photosystem II (PS II) in higher plants. Protein-protein interactions among the structural components of the photosystem will be studied using biochemical and molecular approaches. First, the interaction of the manganese-stabilizing protein with the 24 kDa protein will be examined. Chemical modification of the manganese-stabilizing protein when it is associated with PS II membranes dramatically affects the ability of the 24 kDa protein to bind to PS II. It will determined if the binding inhibition of the 24 kDa protein is due to charge neutralization or steric interference. The location of the modified residues will be mapped by mass spectrometry. Second, the effects of chemical modification on the 24 and 17 kDa proteins with respect to protein binding and function will be determined and the location of the modified residues will be mapped. Third, the role of the observed heterogeneity in the extrinsic subunits of PS II with respect to structure and function of the photosystem will be examined. In Arabidopsis, each of the extrinsic proteins of PS II are encoded by two genes. T-DNA insertion lines, which are available for a number of these genes will be examined. For genes, which have no T-DNA insertional lines available (psbO-2 and psbP-1), RNAi constructs will be introduced to suppress the expression of these gene products. The phenotypes of these plants will be examined with respect to PS II function, assembly and stability under control and stress conditions. In addition to this genetic approach, biochemical reconstitution studies examining the function of the two psbO proteins will be performed. These studies will provide important new information concerning the interactions of the extrinsic components of PS II with the intrinsic components of the photosystem and with each other. Additionally, an understanding of the role of extrinsic subunit heterogeneity may provide insights into regulatory "fine tuning" of the photosynthesis. Finally, the Arabidopsis mutants, which we will generate will provide a resource for examining the role of the extrinsic subunits in vivo.Broader Impacts: This project will involve training at different levels. It is anticipated that several undergraduate students will be involved in research related to this project. These students will attend scientific meetings, presenting their research and ultimately publish their results. The graduate students, postdoctoral researchers and research associates are all intimately involved in the training of these undergraduate researchers. Additionally, the project will continue a long-standing association with Dr. Cindy Putnam-Evans at East Carolina University, a female faculty member in a non-Ph.D.-granting department.