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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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Shapiro, Bert I
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University of Virginia
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Sarver, Jessica L; Zhang, Michael; Liu, Lishan et al. (2018) A Dynamic Protein-Protein Coupling between the TonB-Dependent Transporter FhuA and TonB. Biochemistry 57:1045-1053
Sikora, Arthur; Joseph, Benesh; Matson, Morgan et al. (2016) Allosteric Signaling Is Bidirectional in an Outer-Membrane Transport Protein. Biophys J 111:1908-1918
Joseph, Benesh; Sikora, Arthur; Cafiso, David S (2016) Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR. J Am Chem Soc 138:1844-7
Joseph, Benesh; Sikora, Arthur; Bordignon, Enrica et al. (2015) Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy. Angew Chem Int Ed Engl 54:6196-9
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Cafiso, David S (2014) Identifying and quantitating conformational exchange in membrane proteins using site-directed spin labeling. Acc Chem Res 47:3102-9
Freed, Daniel M; Lukasik, Stephen M; Sikora, Arthur et al. (2013) Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex. Biochemistry 52:2638-48
Regan, Michael C; Horanyi, Peter S; Pryor Jr, Edward E et al. (2013) Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited. Proc Natl Acad Sci U S A 110:13374-9
Flores Jiménez, Ricardo H; Cafiso, David S (2012) The N-terminal domain of a TonB-dependent transporter undergoes a reversible stepwise denaturation. Biochemistry 51:3642-50
Cafiso, David S (2012) Taking the pulse of protein interactions by EPR spectroscopy. Biophys J 103:2047-8

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