Molecular Mechanisms of Membrane Transport

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DESCRIPTION (provided by applicant): Active membrane transport is a critical process for normal cell metabolism, including the maintenance of ion gradients, osmotic balance, action potentials and apoptosis. The proposed work will focus on key questions regarding mechanisms of nutrient uptake in Escherichia coli and other Gram negative bacteria. In E. coli, rare nutrients are sequestered by specific outer membrane proteins that derive energy by coupling to the transperiplasmic protein TonB. These include BtuB, which is responsible for vitamin B12 transport, and FhuA, FecA and FepA, which are responsible for the transport of various forms of chelated iron. High resolution crystallographic models for each of these outer membrane proteins have been obtained; however, the molecular details of the transport machinery remain unclear. The proposed work will utilize site-directed spin labeling and EPR spectroscopy to test models for the molecular mechanisms of TonB-dependent transport in BtuB, and determine the mechanisms by which the transporter-TonB interaction is regulated. The mechanisms of transmembrane signaling resulting from substrate and colicin binding will be examined. Finally, because of the critical need for membrane protein structural biology, the backbone dynamics and structure of beta-barrel motifs, such as BtuB, will be compared in membrane and membrane mimetic systems. In addition to providing fundamental information on membrane proteins and transport, these systems are important to understand for several reasons. TonB-dependent transport provides a model for reversible and regulated protein-protein interactions, macromolecular assembly and transmembrane signal transduction. TonB-dependent transport is also unique to bacteria. Bacteria that are involved in many serious pathologies, such as meningitis, depend upon TonB transport for their success. As a result, understanding TonB transport may lead to the development of new classes of antibiotics that inhibit its function.