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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM035215-25A1
Application #
8761840
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Virginia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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 Jimenez, Ricardo H; Cafiso, David S (2012) The N-terminal domain of a TonB-dependent transporter undergoes a reversible stepwise denaturation. Biochemistry 51:3642-50
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
Freed, Daniel M; Khan, Ali K; Horanyi, Peter S et al. (2011) Molecular origin of electron paramagnetic resonance line shapes on *-barrel membrane proteins: the local solvation environment modulates spin-label configuration. Biochemistry 50:8792-803
Jimenez, 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
Flores Jimenez, Ricardo H; Do Cao, Marie-Ange; Kim, Miyeon et al. (2010) Osmolytes modulate conformational exchange in solvent-exposed regions of membrane proteins. Protein Sci 19:269-78
Do Cao, Marie-Ange; Crouzy, Serge; Kim, Miyeon et al. (2009) Probing the conformation of the resting state of a bacterial multidrug ABC transporter, BmrA, by a site-directed spin labeling approach. Protein Sci 18:1507-20
Xu, Qi; Kim, Miyeon; Ho, K W David et al. (2008) Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein. Biophys J 95:2849-58
Kim, Miyeon; Xu, Qi; Murray, David et al. (2008) Solutes alter the conformation of the ligand binding loops in outer membrane transporters. Biochemistry 47:670-9

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