Energy Transduction Between Membranes

描述（由申请人提供）：生物系统中的一次能量转导来自两个耦合源。氧化磷酸化（和底物磷酸化）在反应物和产物在物理和空间上耦合的反应中最终产生离子动力和细胞质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，通过传统方式产生的能量（细胞质膜原动力）可以被转导到外膜的蛋白质中，以便将营养物质主动运输到质周空间。这一系统似乎构成了一种新的生物学范式。使用E. coil作为模型系统，已经清楚地表明，在细胞质和外膜上有几种蛋白质参与了这一过程。在这个过程中的核心角色，TonB，被假设携带构象约束的势能，并在两个膜之间来回穿梭以获取并传递它。在细胞质膜中，ExbB和ExbD被假设为将Ton B转化为其能量形式，并提供运行航天飞机的能量。外膜中有TonB可以与之对接的蛋白质，也有能量被传递到的外膜转运蛋白。在这个建议中，进一步了解能量转导机制的分子细节。具体来说，我们将1)确定TonB氨基和羧基末端的功能；2)研究穿梭的能量来源；3)继续确定ExbB和ExbD的作用；4)基于colicin B的结构启动研究，以表征TonB依赖性colicin感染的阶段，从外膜开始。

DESCRIPTION (provided by applicant): Primary energy transduction in biological systems is derived from two coupled sources. Oxidative phosphorylation (and substrate phosphorylation) ultimately generate both an ion motive force and cytoplasmic 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 by conventional means (cytoplasmic membrane protonmotive force) can be transduced to transport proteins in the outer membrane for active transport of nutrients into the periplasmic space. This system appears to constitute a new biological paradigm. Using E. coil as a model system, it has become clear that several proteins are involved in this process, at both the cytoplasmic and outer membranes. The central player in this process, TonB, is hypothesized to carry conformationally constrained potential energy, and shuttle back and forth between the two membranes to obtain and then deliver it. In the cytoplasmic membrane ExbB and ExbD are hypothesized to convert Ton B to its energized form and to provide the energy that runs the shuttle. In the outer membrane are proteins with which TonB may dock as well as the outer membrane transporters to which the energy is delivered. In this proposal a further understanding of the molecular details of the energy transduction mechanism are sought. Specifically, we will 1) define the functions of the TonB amino and carboxy termini 2) investigate the energy source for the shuttle 3) continue to define the roles of ExbB and ExbD, and 4) initiate studies, based on the structure of colicin B, to characterize the stages of TonB-dependent colicin infection, starting at the outer membrane.