Organization of Arabinose Transport Proteins

该研究计划的这一目标旨在确定和了解高亲和力L-阿拉伯糖运输系统中三个组成部分(Araf、Arag和Arah)的作用以及它们相互作用的方式，以影响L-阿拉伯糖从环境中高效积累并用于代谢利用。通过饱和突变鉴定，阿拉伯糖结合蛋白(ARAF产物)裂解表面上的残基参与膜复合体的相互作用，将通过定点突变单独进行修饰，以确定这种相互作用的化学要求。膜复合体的组成成分(Arag和Arah)的组织将通过使用以正确和相反方向制备的囊泡选择性地修饰暴露在细胞膜内外表面的残基来确定。放射性标记的修饰蛋白将通过十二烷基硫酸钠-PAGE和放射自显影进行可视化。从生化修饰实验中获得的信息将与TnPhoA插入的性质相关联，以确定膜环境中单个成分的取向。Arag蛋白的作用将通过将放射性标记的核苷酸紫外线交联到假定的三磷酸腺苷结合位点和确定的偏好位点(S)中的一个或两个来实现。结合蛋白运输系统提供了一个理想的模型来考虑细胞与细胞膜外环境的通信方式，表面受体是如何组织的，细胞如何在脂膜中产生毛孔来适应亲水性分子的运输，细胞如何将蛋白质引导到细胞内、膜内或细胞外的特定位置，细胞如何利用能量来影响底物积累。对这类系统的进一步研究只会增加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.