Molecular Mechanisms of Membrane Transport

描述（由申请人提供）：活性膜转运是正常细胞代谢的关键过程，包括离子梯度、渗透平衡、动作电位和凋亡的维持。提出的工作将集中在大肠杆菌和其他革兰氏阴性菌的营养摄取机制的关键问题上。在大肠杆菌中，罕见的营养物质被特定的外膜蛋白隔离，这些外膜蛋白通过与外质蛋白TonB偶联获得能量。其中包括负责维生素B12运输的BtuB，以及负责运输各种形式螯合铁的FhuA， FecA和FepA。获得了这些外膜蛋白的高分辨率晶体学模型；然而，运输机制的分子细节仍不清楚。这项工作将利用位点定向自旋标记和EPR光谱来测试BtuB中tonb依赖转运的分子机制模型，并确定转运体- tonb相互作用的调节机制。由底物和粘菌素结合产生的跨膜信号传导机制将被研究。最后，由于膜蛋白结构生物学的迫切需要，β -桶基序（如BtuB）的骨干动力学和结构将在膜和膜模拟系统中进行比较。除了提供关于膜蛋白和转运的基本信息外，这些系统还有几个重要的理解原因。tonb依赖性转运为可逆和受调节的蛋白质相互作用、大分子组装和跨膜信号转导提供了一个模型。依赖吨的运输也是细菌所特有的。与许多严重疾病（如脑膜炎）有关的细菌依赖于TonB的转运才能成功。因此，了解TonB转运可能会导致开发抑制其功能的新型抗生素。

DESCRIPTION (provided by applicant): Active membrane transport is a critical process for normal cell metabolism, including the maintenance of ion gradients, osmotic balance, action potentials and apoptosis. The proposed work will focus on key questions regarding mechanisms of nutrient uptake in Escherichia coli and other Gram negative bacteria. In E. coli, rare nutrients are sequestered by specific outer membrane proteins that derive energy by coupling to the transperiplasmic protein TonB. These include BtuB, which is responsible for vitamin B12 transport, and FhuA, FecA and FepA, which are responsible for the transport of various forms of chelated iron. High resolution crystallographic models for each of these outer membrane proteins have been obtained; however, the molecular details of the transport machinery remain unclear. The proposed work will utilize site-directed spin labeling and EPR spectroscopy to test models for the molecular mechanisms of TonB-dependent transport in BtuB, and determine the mechanisms by which the transporter-TonB interaction is regulated. The mechanisms of transmembrane signaling resulting from substrate and colicin binding will be examined. Finally, because of the critical need for membrane protein structural biology, the backbone dynamics and structure of beta-barrel motifs, such as BtuB, will be compared in membrane and membrane mimetic systems. In addition to providing fundamental information on membrane proteins and transport, these systems are important to understand for several reasons. TonB-dependent transport provides a model for reversible and regulated protein-protein interactions, macromolecular assembly and transmembrane signal transduction. TonB-dependent transport is also unique to bacteria. Bacteria that are involved in many serious pathologies, such as meningitis, depend upon TonB transport for their success. As a result, understanding TonB transport may lead to the development of new classes of antibiotics that inhibit its function.