MOLECULAR ORGANIZATION OF A MEMBRANE PROTEIN COMPLEX

我们的研究目标是：1）确定结构 光系统II（PS II）核心复合物多肽的拓扑结构， 2)来描述 亚基和3）产生残基的位点特异性突变 其被预测为协调电子传输组件， 设置氧化还原电位并调节电荷转移动力学。 最近，我们已经开发出快速定位 跨膜多肽跨度的位置和方向， 针对合成肽产生的位点特异性抗体。 的 对应于亲水序列合成肽抗原 包围每个潜在的跨膜跨度。 通过分析 抗体结合模式由内而外和右侧外 我们决定膜的位置和方向 跨膜跨度。 我们的调查结果显示 跨膜区的数量和方向 PS Ⅱ复合物的D1蛋白与此不一致 通过疏水性图预测。 此外，显然 D1蛋白在结构上与L亚基同源， 绿色红单胞菌光合反应中心。 这些结果的含义是，D1蛋白协调 PS II反应中心初级电子受体和供体， 异二聚体复合物（D1和D2）类似于R. viridis L和 M亚单位。 利用上述免疫学技术 我们将继续分析PS II的蛋白质拓扑结构 D2、43和47 kd叶绿素复合多肽 结合蛋白和细胞色素b559亚基。 这些研究 将通过残基的定点诱变来补充 结合电子供体和受体， PS II反应中心光化学。 这些调查人员将 确定复杂的膜蛋白结构是否可以 有效地解剖使用各种免疫和 重组DNA技术。 我们建议这项研究将 在许多膜蛋白系统中具有广泛的应用。

Our research objectives are: 1) to determine the structural topology of the photosystem II (PS II) core complex polypeptides, 2) to characterize the structural relationships between the subunits and 3) to generate site specific mutations of residues which are predicted to coordinate electron transport components, set redox potentials and regulate charge transfer kinetics. Recently, we have developed methods to rapidly locate the position and orientation of transmembrane polypeptide spans using site specific antibodies generated against synthetic peptides. The synthetic peptide antigens correspond to hydrophilic sequences bracketing each potential transmembrane span. By analysis of the antibody binding patterns to inside out and right side out membranes we determine the position and orientation of transmembrane spans. The results of our investigations indicated that the numbers and orientations of transmembrane spans in the D1 protein of the PS II complex did not correspond with that predicted by hydrophobicity plots. In addition it was apparent that the D1 protein was structurally homologous to the L subunit of the Rhodopsuedomonas viridis photosynthetic reaction center. The implication of these results is that the D1 protein coordinates the PS II reaction center primary electron acceptors and donors in a heterodimer complex (D1 and D2) similar to the R. viridis L and M subunits. Using the aforementioned immunological techniques we will continue our analyses of the protein topology of the PS II complex polypeptides including the D2, 43 and 47 kd chlorophyll binding proteins and the cytochrome b559 subunits. These studies will be supplemented by site directed mutagenesis of residues which bind the electron donors and acceptors and which regulate PS II reaction center photochemistry. These investigators will determine whether complex membrane protein structures can be effectively dissected using a variety of immunological and recombinant DNA techniques. We propose that this research will have broad application in many membrane protein systems.