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 bacterial and are critical to the success of many bacterial pathogens, such as the bacteria that result in meningitis, cholera and pertussis. Because they are unique to bacteria, these transporters are a logical 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 mechanisms by which transport takes place is unclear. The proposed work will utilize site-directed spin labeling and EPR spectroscopy to test models for the molecular mechanisms of TonB-dependent transport, determine the mechanisms of transmembrane signaling and determine the mechanisms by which the transporter-TonB interaction is regulated. Finally, the structure and dynamics of these transporters (which are based upon 2-barrels) are influenced by both solute and lipid environment. Because of the critical need to generate and interpret high-resolution structural models of membrane proteins, the proposed work will also quantitate the influence of solutes and lipid environment on the structure of this class of membrane proteins.
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 that can inhibit bacterial growth.
|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|
Showing the most recent 10 out of 43 publications