Active membrane transport is central to many cell processes, including the acquisition of nutrients, the establishment of ion-gradients, the regulation of osmotic balance, ATP synthesis, and apoptosis. The proposed work will address key questions regarding the mechanisms of nutrient uptake in Escherichia coli, and it will address questions regarding the structure and organization of these proteins in the bacterial outer-membrane. In E. coli, a range of nutrients are transported 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 they are critical to the proper functioning of the human microbiome where they are responsible for the acquisition and initial processing of some carbohydrates. They are also essential for the success of many bacterial pathogens, such as those that result in meningitis, cholera, pertussis and dysentery; and because TonB-dependent transport is unique to bacteria, it is thought to be a target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for approximately two dozen TonB- dependent transporters; however, the mechanism by which transport takes place is unclear. One difficulty is that structural and biophysical studies in-vitro have never been made on TonB- dependent transport proteins that are known to be active. The proposed work will test models for the molecular mechanisms of transport and examine structural states that are only observed in cells. The role of lipopolysaccharide in altering transport protein structure will also be examined. The proposed work will employ site-directed spin labeling and EPR spectroscopy where novel approaches have been developed to perform pulse experiments, such as double electron-electron resonance, in intact E. coli. In the outer-membrane, proteins are sequestered into domains or islands, which are thought to drive the turnover of outer-membrane proteins in bacteria. EPR will be used in E. coli to characterize the protein-protein interactions that drive domain formation and define the supramolecular structure of the outer-membrane.