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)核心复合多肽的拓扑结构， 2)刻画 亚基和3)产生残基的位点特异性突变 它们被预测为协调电子传输组件， 设置氧化还原电位，调节电荷转移动力学。 最近，我们开发了一些方法来快速定位 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。这个 合成肽抗原对应于亲水序列 包括每一个潜在的跨膜跨度。通过对该模型的分析 抗体结合模式由内向外和右侧向外 我们决定膜的位置和方向 跨膜跨度。我们的调查结果表明 跨膜跨膜的数量和方向 PS II复合体的D1蛋白与之不一致 通过疏水性图预测。此外，很明显 D1蛋白在结构上与L亚基同源 绿色红假单胞菌光合作用反应中心。 这些结果的含义是d1蛋白与 PS II反应中心初级电子受体和供体 异二聚体复合体(D_1和D_2)，类似于绿色弧菌L和 M亚基。使用上述免疫学技术 我们将继续分析PS II的蛋白质拓扑结构 包括D2、43和47kd叶绿素在内的复合多肽 结合蛋白和细胞色素b559亚基。这些研究 将通过残基的定点突变来补充 它结合了电子供体和受体，并调节了 PS II反应中心光化学。这些调查人员将 确定复合膜蛋白结构是否可以 有效地解剖了使用各种免疫学和 重组DNA技术。我们建议这项研究将 在许多膜蛋白体系中有着广泛的应用。

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.