Energy Transduction Between Membranes

描述（由申请人提供）：生物系统中的初级能量转换来自两个耦合源。氧化磷酸化（和底物磷酸化）最终在反应物和产物物理和空间耦合的反应中产生离子动力和细胞质ATP。这两种能量来源都用于驱动营养物质穿过细胞质膜的主动运输。革兰氏阴性菌的外膜存在一个特殊的问题：它们没有能量，对大于600 Da的分子是不渗透的。为了获得重要的营养，这些生物体已经发展出一种复杂的系统，通过该系统，由常规方法（细胞质膜质子动力）产生的能量可以被转换以在外膜中转运蛋白质，从而将营养物主动转运到周质空间中。这个系统似乎构成了一个新的生物学范式。使用大肠coil作为模型系统，已经清楚的是，在细胞质和外膜上，几种蛋白质参与了这一过程。在这个过程中的核心球员，TonB，被假设为携带构象约束的势能，并穿梭在两个膜之间，以获得，然后传递它。在细胞质膜Exb B和ExbD被假设为转换Ton B到它的能量形式，并提供运行穿梭的能量。在外膜中是TonB可以与之对接的蛋白质以及能量被递送到的外膜转运蛋白。在这个建议中，寻求进一步了解的能量转导机制的分子细节。具体而言，我们将1）定义TonB B氨基和羧基末端的功能2）研究穿梭的能量来源3）继续定义Exb B和ExbD的作用，以及4）基于大肠杆菌素B的结构启动研究，以表征TonB依赖性大肠杆菌素感染的阶段，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.