Energy Transduction Between Membranes

描述(申请人提供)：生物系统中的初级能量转导来自两个耦合的源。在反应物和产物在物理和空间上耦合的反应中，氧化磷酸化(和底物磷酸化)最终产生离子动力和细胞质ATP。这两种能源都被用来推动营养物质在细胞质膜上的主动运输。革兰氏阴性细菌的外膜存在一个特殊的问题：它们不带电，而且它们不适合大于600Da的分子。为了获得重要的营养物质，这些生物体发展了一种复杂的系统，通过这种系统，通过传统方式产生的能量(细胞膜质子动力)可以被传递到外膜中的蛋白质，从而主动地将营养物质运输到周质空间。这一系统似乎构成了一种新的生物学范式。以大肠杆菌为模型系统，很明显，在细胞质和外膜上都有几种蛋白质参与这一过程。这一过程中的中心角色TonB被假设携带构象受限的势能，并在两层膜之间来回穿梭以获得并传递势能。在细胞质膜上，ExbB和ExbD被认为是将Ton B转化为其带电的形式，并提供运行航天飞机的能量。在外膜中有TonB可能与之对接的蛋白质，以及能量被输送到的外膜转运体。在这一提议中，寻求对能量转导机制的分子细节的进一步了解。具体地说，我们将1)定义TonB氨基和羧基末端的功能2)研究穿梭的能源3)继续定义ExbB和ExbD的作用，以及4)基于Colicin B的结构，从外膜开始研究TonB依赖的Colicin感染的阶段。

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.