TonB-dependent transport across the outer membrane

 描述(申请人提供)：革兰氏阴性细菌的外膜是许多重要营养物质扩散的障碍，如铁载体进入周质空间。为了绕过这些限制，具有非常高亲和力的活性转运体位于外膜。然而，外膜缺乏足够离子梯度的常规能源或获得三磷酸腺苷。相反，通过三种完整的细胞质膜蛋白TonB-ExbB-ExbD从细胞质膜质子动力中传递通过外膜主动运输的能量。ExbB/D采集质子动力并将其传递给TonB，TonB直接接触外膜运输器。能量转导到外膜转运体的确切机制尚不清楚。我们的长期目标是以大肠杆菌为模型系统，了解细胞质膜和外膜之间TonB依赖的能量转导机制。由于其在铁获取中的作用，TonB系统是许多革兰氏阴性病原体的毒力因子。我们的长期目标是了解大肠杆菌细胞质和外膜之间依赖TonB的能量转导的机制。过去的体外研究已经鉴定了TonB周质结构域(缺乏必要的跨膜结构域)与纯化的转运蛋白的结合。到目前为止，实际的运输还没有得到演示。我们将确定活性全长TonB和外膜转运蛋白FepA在体内转运过程中发生的残基特异性能量依赖的相互作用。这些知识将被用来评估FepA和活性全长TonB之间的体外相互作用。我们还将识别TonB系统中的未知蛋白质，并表征它们所起的作用。

 DESCRIPTION (provided by applicant): The outer membranes of Gram-negative bacteria are barriers to diffusion of many important nutrients such as iron-siderophores into the periplasmic space. To circumvent these limitations, active transporters with very high affinities for their transport ligands are located in the outer membrane. However the outer membrane lacks conventional energy resources of sufficient ion gradients or access to ATP. Instead, energy for active transport across the outer membrane is transduced from the cytoplasmic membrane proton motive force by three integral cytoplasmic membrane proteins, TonB-ExbB-ExbD. ExbB/D harvest the proton motive force and transmit it to TonB, which directly contacts the outer membrane transporter. The exact mechanism of energy transduction to the outer membrane transporter is unknown. Our long- term goal is to understand the mechanism of TonB-dependent energy transduction between the cytoplasmic and outer membranes using Escherichia coli as the model system. Because of the role it plays in iron acquisition the TonB system is a virulence factor for many Gram-negative pathogens. Our long-term goal is to understand the mechanism of TonB-dependent energy transduction between the cytoplasmic and outer membranes of Escherichia coli. Past in vitro studies have characterized binding of TonB periplasmic domains (lacking the essential transmembrane domain) to purified transporters. To date, actual transport has not been demonstrated. We will identify the residue-specific energy-dependent interactions that occur between active full-length TonB and the outer membrane transporter FepA during transport in vivo. This knowledge will be used to evaluate in vitro interactions between FepA and active full-length TonB. We will also identify unknown proteins in the TonB system and characterize the role they play.