Organization of Arabinose Transport Proteins

该研究计划的目标是定义和 了解三个组件（araF，araG， 和araH）的高亲和力L-阿拉伯糖转运系统， 它们相互作用的方式， 从环境中积累L-阿拉伯糖用于代谢 利用率 阿拉伯糖裂面残基 结合蛋白（araF的产物）参与相互作用， 通过饱和诱变鉴定的膜复合物将 通过定点诱变单独修饰， 建立这种相互作用的化学要求。 的 膜复合物的组分（araG和 araH）将通过残基的选择性修饰来确定 暴露于细胞质膜的内外表面 使用以正确和倒置方向制备的囊泡。 将通过SDS PAGE观察放射性标记的修饰蛋白 和放射自显影。 从生物化学中获得的信息 改性实验将与性能相关 的TnphoA插入，以建立 膜环境中的单个组件。 的作用 araG蛋白将通过UV交联 放射性标记的核苷酸进入一个或两个推定的ATP 结合位点和识别的优选位点。 结合蛋白转运系统提供了一个理想的模型， 考虑细胞与 细胞质膜外的环境， 受体是有组织的，细胞如何在脂质中产生孔 膜，以适应过境的亲水性分子，如何 细胞将蛋白质引导到细胞中的特定位置， 细胞膜或细胞外，细胞如何利用能量来影响 底物积累 进一步审议此类系统 只会加深我们对细胞过程的理解

This goal of the research program proposes to define and understand the role of each of the three components (araF, araG, and araH) of the high-affinity L-arabinose transport system and the manner in which they interact to effect the efficient accumulation of L-arabinose from the environment for metabolic utilization. Residues on the cleft surface of the arabinose binding protein ( product of araF) that engage in interactions at the membrane complex as identified by saturation mutagenesis will be individually modified by site directed mutagenesis to establish the chemical requirements of that interaction. The organization of the components of the membrane complex (araG and araH) will be determined by selective modification of residues exposed to the inner outer surfaces of the cytoplasmic membrane using vesicles prepared in the correct and inverted orientation. Radiolabelled modified proteins will be visualized by SDS PAGE and radioautography. Information obtained from the biochemical modification experiments will be correlated with the properties of TnphoA insertions to establish the orientation of the individual components in the membrane environment. The role of the araG protein will be pursued by UV crosslinking of radiolabelled nucleotides into one or both of the putative ATP binding sites and the site(s) of preference identified. Binding protein transport systems provide an ideal model to consider the manner by which cells communicate with the environment outside the cytoplasmic membrane, how surface receptors are organized, how cells generate pores in lipid membranes to accomodate transit of hydrophilic molecules, how cells direct proteins to specific locations in the cell, in the membrane, or outside the cell, how cells utilize energy to effect substrate accumulation. Further consideration of such systems can only add to our understanding of cellular processes.