ENERGY TRANSDUCTION BETWEEN MEMBRANES

生物系统中的初级能量转导来源于两个耦合源。氧化磷酸化（和底物水平磷酸化）在反应物和产物物理和空间耦合的反应中最终产生离子动力和ATP。这两种能量来源都用来驱动营养物质在细胞质膜上的主动运输。革兰氏阴性菌的外膜有一个特殊的问题：它们没有能量，不适合大于600 Da的分子。为了获得重要的营养物质，这些生物已经发展出一种复杂的系统，通过这种系统，细胞质膜上产生的能量（以质子动力的形式）可以被转导成外膜上的蛋白质，从而将这些重要的营养物质主动运输到两层膜之间的质周空间。利用大肠杆菌作为模型系统，很明显有几种蛋白质参与了这一过程。能量转导系统的核心是TonB蛋白，它可以与细胞质和外膜上的蛋白质接触。两种细胞质膜蛋白ExbB和ExbD似乎需要为TonB提供能量，然后在能量转导后再循环。存在多种依赖tonb的外膜受体。跨越空间的能量传导机制——可能构成一种新的范式——尚未得到很好的理解。目前的数据表明，TonB在细胞质和外膜之间来回穿梭。ExbB和ExbD在这一过程中所起的作用尚不清楚。此外，有证据表明，其他未表征的蛋白质参与了这一过程。本提案的目的是针对这些领域进行调查。具体来说，我们将1)确定这些能量转导复合物的组成和化学计量，2)确定TonB在膜间的物理穿梭是否是能量转导的基本特征，3)阐明ExbB和ExbD的作用，4)定义TonB与外膜受体和其他外膜组分的主要相互作用，5)确定TonB物理储存能量的机制。

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.