Protein-Protein Interactions in Photosystem II

9604339 Bricker第1部分-技术这项研究将进一步阐明光系统II中蛋白质的结构组织。将结合生物化学和分子工具研究光系统各组成部分之间的蛋白质-蛋白质和蛋白质-辅因子相互作用。首先，位点特异性蛋白质标记试剂NHS-生物素将被用来探测光系统外部蛋白质上的相互作用结构域。将对一些生化分解的光系统II复合体和非溶液中的外源蛋白进行标记。33 kDa、24 kDa和17 kDa外源蛋白的修饰结构域将通过亲和纯化、蛋白酶消化和基质辅助激光解吸/电离质谱仪相结合的方法进行鉴定。其次，将对CP 7突变体W167S和RR384385EE的自发和化学诱导回复进行抑制分析。基因间抑制基因将被识别、定位和测序，以确定与CP 47蛋白中这些位置相互作用的蛋白质的身份。第三，使用非特定化学蛋白酶Fe-EDTA的蛋白质足迹将被用来描绘光系统内的相互作用。处于不同生化分解状态的光系统II复合体将受到化学蛋白酶的处理。一些光系统II组分(CP 47、D 1和33 kDa外源蛋白)上的保护域将通过“Western blotting”和N-末端和C-末端特异性抗体试剂探测来识别。这些实验应该提供关于这些蛋白质成分上相互作用区域的相对较高的分辨率信息。第二部分-膜蛋白复合体的功能性质和结构组织的非技术阐明是当前生物化学研究的中心目标之一。生物膜几乎涉及细胞组织和活动的方方面面。膜功能最令人感兴趣的方面之一是它在光合作用生物体中调节能量传递的作用。光能是最剧烈的物理过程--聚变的产物，它被转化为生物能量当量，供光合作用细胞利用。光合作用过程提供碳水化合物，碳水化合物位于几乎所有食物链的底部，并作为副产品提供高等生物利用的所有大气氧气。这个项目研究了参与放氧的蛋白质之间的结构相互作用。了解放氧复合体的结构将为这一重要的膜蛋白复合体的功能提供新的见解。

9604339 Bricker Part 1-Technical This research will further elucidate the structural organization of proteins within Photosystem II. Protein-protein and protein-cofactor interactions among the components of the photosytem will be studied using a combination of biochemical and molecular tools. First, the site-specific protein-labeling reagents NHS-biotin will be used o probe interacting domains on the extrinsic proteins of the photosystem. Labeling will be performed on a number of biochemically resolved Photosystem II complexes and on the extrinsic proteins free in solution The modified domains on the 33 kDa, 24 and 17 kDa extrinsic proteins will be identified by a combination of affinity purification, protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. Second, suppressor analysis will be performed on spontaneous and chemically induced revertants of the CP 7 mutants W167S and RR384385EE. Intergenic suppressors will be identified, mapped and sequenced to determine the identity of proteins interacting with these location in the CP 47 protein. Third, protein footprinting using the non-specific chemical protease Fe-EDTA will be used to map interactions within the photosystem. Photosystem II complexes in various states of biochemical resolution will be subjected to chemical protease treatment. Protected domains on a number of Photosystem II components (CP 47, D1 and the 33 kDa extrinsic protein) will be identified by "Western blotting" and probing with N-and C-terminal specific antibody reagents. These experiments should provide relatively high resolution information concerning interacting domains on these protein components. Part 2-Non Technical Elucidation of the functional properties and structural organization of membrane protein complexes is one of the central objectives of current biochemical investigation. Biological membranes are involved in virtually every aspect of cellular organization and activity. One of the most intriguing aspects of membrane function is its role in the mediation of energy transduction in photosynthetic organisms. Light energy, which is the product of a most violent physical process, fusion, is transformed into biological energy equivalents utilized by the photosynthetic cell. The photosynthetic process provides both the carbohydrate which lies at the base of virtually all food chains and, as a byproduct, all of the atmospheric oxygen utilized by higher organisms. This project investigates the structural interactions between the proteins involved in oxygen evolution. An understanding of the structure of the oxygen evolving complex will yield new insights into the how this important membrane protein complex functions.