STRUCTURE AND ORGANIZATION OF THE PHOTOSYSTEM I COMPLEX

到目前为止，很少有膜蛋白的结构被确定。 在原子分辨率下。我们计划研究分子组织。 光系统I并以原子分辨率确定其结构。 光系统I，光依赖的叶绿体蛋白-铁氧还蛋白氧化还原酶， 是光合成中的一种异多聚体色素-蛋白质复合体 叶绿体和蓝藻的膜。它至少包含11个 蛋白质、100个叶绿素、β-胡萝卜素和一系列电子 转运中心。光系统I是一种典型的膜蛋白复合体。 此外，还具有稳定性好、含量丰富、易于提纯等特点。因此，光系统I是 一种适用于膜蛋白结构分析的模型体系 复合体。我们已经开始了一项全面的项目，旨在澄清 嗜热蓝藻光系统I的结构 叶片长管藻。我们这个项目的主要目标是： (1)光系统I组织的地形分析。我们将确定 暴露在复合体表面的蛋白质结构域或 参与复合体内蛋白质之间的相互作用。国民健康保险制度- 生物素化、蛋白酶可及性和化学交联性研究 将会被使用。这些方法生成的信息将用于 确定复合体中各个亚基的结构。这些数据将 还帮助正确识别结构组件 X射线结晶学。 (2)用于结构分析的较简单系统的生成。我们会 克隆和鉴定光系统I蛋白的一级结构 对相应基因进行测序。我们将过度生产外围设备 光系统I亚基在大肠杆菌中的表达及结构分析 结晶学。我们还将产生缺乏亚基的突变体 PSAF和PSAL蛋白，从而设计出一种更简单的复合体， 更亲水，但仍有功能。 (3)X射线衍射分析：我们将结晶完整，更简单 光系统L复合体以及过表达的蛋白质。我们计划 通过X-射线衍射谱分析确定其结构。 最后，我们将整合生化和X射线结晶学数据以 提出了光系统I的详细结构模型。

Until now, structures of very few membrane-proteins have been determined at atomic resolution. We plan to study molecular organization of photosystem I and determine its structure at atomic resolution. Photosystem I, the light-dependent plastocyanin-ferredoxin oxidoreductase, is a heteromultimeric pigment-protein complex in the photo synthetic membranes of chloroplasts and cyanobacteria. It contains at least eleven proteins, 100 chlorophylls, beta-carotenes and a series of electron transfer centers. Photosystem I is a typical membrane-protein complex. Moreover, it is stable, abundant and easy to purify. Thus photosystem I is a suitable model system for structural analysis of membrane-protein complexes. We have embarked on a comprehensive project aimed to elucidate structure of photosystem I from the thermophilic cyanobacterium Mastigocladus laminosus. Our main objectives for this project are: (1) Topographical analysis of photosystem I organization. We will identify protein domains that are exposed on the surface of the complex or are involved in protein-protein interactions within the complex. NHS- biotinylation, protease accessibility, and chemical cross-linking studies will be used. The information generated by these methods will be used to orient structures of individual subunits in the complex. These data will also assist in correct identification of structural components revealed by X-ray crystallography. (2) Generation of simpler systems for structural analysis. We will determine primary structure of photosystem I proteins by cloning and sequencing the corresponding genes. We will overproduce peripheral subunits of photosystem I in E. coli and elucidate their structure by crystallography. We will also generate subunit-deficient mutants lacking the PsaF and PsaL proteins, thus engineering a complex that is simpler, more hydrophilic, but still functional. (3) X-ray diffraction analysis: We will crystallize entire and simpler photosystem l complexes as well as the overexpressed proteins. We plan to determine their structures from analysis of X-ray diffraction patterns. Finally we will integrate biochemical and X-ray crystallographic data to propose model for the detailed structure of photosystem I.