ENERGY TRANSDUCTION BETWEEN MEMBRANES

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Primary energy transduction in biological systems is derived from two coupled sources. Oxidative phosphorylation (and substrate level phosphorylation) ultimately generate both an ion motive force and ATP in reactions where the reactants and products are physically and spatially coupled. Both of these energy sources are used to drive active transport of nutrients across the cytoplasmic membrane. The outer membranes of Gram-negative bacteria present a special problem: they are unenergized and they are impermeant to molecules of greater than 600 Da. In order to obtain vital nutrients, these organisms have developed a sophisticated system whereby energy generated at the cytoplasmic membrane (in the form of proton motive force) can be transduced to proteins in the outer membrane for active transport of these vital nutrients into the periplasmic space between the two membranes. Using E. coli as a model system, it has become clear that several proteins are involved in this process. The core of the energy transducing system is TonB protein, which can contact proteins in both the cytoplasmic and outer membranes. Two cytoplasmic membrane proteins, ExbB and ExbD appear to be required to energize TonB and then recycle it, following energy transduction. A variety of TonB-dependent outer membrane receptors exist. The mechanism of energy transduction across space--which may constitute a new paradigm--is not well understood. Current data suggest that TonB shuttles back and forth between the cytoplasmic and outer membranes. The roles that ExbB and ExbD play in this process are not well understood. Furthermore, there is evidence that additional uncharacterized proteins are involved in this process. The aims of this proposal are directed toward investigating these areas. Specifically, we will 1) resolve the composition and stoichiometry of these energy-transducing complexes, 2) determine if the physical shuttling of TonB between membranes is an essential feature of energy transduction, 3) elucidate the roles of ExbB and ExbD, 4) define the primary interactions of TonB with the outer membrane receptors and other outer membrane components, 5) determine the mechanism by which TonB physically stores energy.