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.

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 that can inhibit bacterial growth.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035215-23
Application #
7994180
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Ainsztein, Alexandra M
Project Start
1985-09-06
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2011-11-30
Support Year
23
Fiscal Year
2011
Total Cost
$285,751
Indirect Cost
Name
University of Virginia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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
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 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
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
Jiménez, 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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