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.
