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 address key questions regarding the 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 inner-membrane protein TonB. These TonB-dependent transporters 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. TonB-dependent transporters are abundant in Gram negative bacteria and are critical to the success of many bacterial pathogens, such as the bacteria that result in meningitis, cholera, pertussis and dysentery. Because they are unique to bacteria, these transporters are a rational target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for a number of TonB-dependent transporters;however, the mechanism by which transport takes place is unclear. The proposed work will determine the mechanisms by which protein-protein interactions are regulated in this system and test models for the molecular mechanisms of transport. Site-directed spin labeling and EPR spectroscopy in combination with high-resolution NMR will be used to examine ligand- induced structural changes, dynamics and conformational exchange within these transporters. The proposal will use novel approaches test for conformational dynamics and map changes in the energy landscape within these membrane proteins. Approaches will be used to increase the population of excited conformational states so that intermediate structures in the transport process may be characterized.
The proposed research will determine the molecular mechanisms by which bacteria transport scarce nutrients across their cell membrane. This transport is critical to the survival of bacteria, and it is essential for the success of many bacterial pathogens, such as the bacteria that cause meningitis, cholera and pertussis. Knowledge of these transport mechanisms will assist with the development of new antibiotics to treat bacterial infection.
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|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|
|Iyalomhe, Osigbemhe; Herrick, Dawn Z; Cafiso, David S et al. (2014) Closure of the cytoplasmic gate formed by TM5 and TM11 during transport in the oxalate/formate exchanger from Oxalobacter formigenes. Biochemistry 53:7735-44|
|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|
|Flores JimÃ©nez, Ricardo H; Cafiso, David S (2012) The N-terminal domain of a TonB-dependent transporter undergoes a reversible stepwise denaturation. Biochemistry 51:3642-50|
|Mokdad, Audrey; Herrick, Dawn Z; Kahn, Ali K et al. (2012) Ligand-induced structural changes in the Escherichia coli ferric citrate transporter reveal modes for regulating protein-protein interactions. J Mol Biol 423:818-30|
|JimÃ©nez, Ricardo H Flores; Freed, Daniel M; Cafiso, David S (2011) Lipid and membrane mimetic environments modulate spin label side chain configuration in the outer membrane protein A. J Phys Chem B 115:14822-30|
|Ellena, Jeffrey F; Lackowicz, Pawel; Mongomery, Hillary et al. (2011) Membrane thickness varies around the circumference of the transmembrane protein BtuB. Biophys J 100:1280-7|
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