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。我们计划研究 光系统I并以原子分辨率确定其结构。 光系统I，光依赖性质体蓝素-铁氧还蛋白氧化还原酶， 是光合作用中的异源多聚体色素蛋白复合物， 叶绿体和蓝细菌的膜。它至少包含11个 蛋白质，100叶绿素，β-胡萝卜素和一系列的电子 转移中心。光系统I是典型的膜蛋白复合体。 此外，它是稳定的，丰富的，易于纯化。因此，光系统I是 一个适用于膜蛋白结构分析的模型系统 配合物我们已经开始了一个全面的项目，旨在阐明 嗜热蓝细菌光系统I的结构 Mastigocladus laminosus.本项目的主要目标是： (1)光系统I组织的地形分析。我们将确定 暴露在复合物表面上或 参与蛋白质间的相互作用。NHS- 生物素化、蛋白酶可及性和化学交联研究 将用于这些方法产生的信息将用于 确定复合体中单个亚基的结构方向。这些数据将 还有助于正确识别结构部件， X射线晶体学 (2)生成用于结构分析的更简单的系统。我们将 通过克隆确定光系统I蛋白的一级结构， 测序相应的基因。我们将过度生产外围设备 光系统I亚基。大肠杆菌，并阐明其结构， 结晶学我们还将产生缺乏亚基的突变体， PsaF和PsaL蛋白，从而工程化一种更简单的复合物， 更亲水，但仍有功能。 (3)X射线衍射分析：我们将结晶完整和更简单 光系统L复合物以及过表达的蛋白质。我们计划 通过X射线衍射图分析确定其结构。 最后，我们将整合生物化学和X射线晶体学数据， 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.