Molecular Mechanisms of Membrane Transport

描述(申请人提供)：主动膜转运是细胞正常代谢的关键过程，包括维持离子梯度、渗透平衡、动作电位和细胞凋亡。这项拟议的工作将解决有关大肠杆菌和其他革兰氏阴性细菌吸收营养的机制的关键问题。在大肠杆菌中，稀有的营养物质被特定的外膜蛋白隔离，这些外膜蛋白通过与内膜蛋白TonB偶联来获得能量。这些依赖TonB的转运蛋白包括BtuB和FhuA、FECA和Fepa，BtuB负责维生素B12的运输，FhuA、FECA和Fepa负责运输各种形式的螯合铁。依赖于TonB的转运蛋白在革兰氏阴性细菌中大量存在，对许多细菌病原体的成功至关重要，如导致脑膜炎、霍乱和百日咳的细菌。因为它们是细菌独有的，所以这些转运蛋白是开发新类别抗生素的合理目标。已经获得了一些依赖于TonB的转运蛋白的高分辨率结晶学模型；然而，发生转运蛋白的机制尚不清楚。这项拟议的工作将利用定点自旋标记和EPR光谱来测试TonB依赖的转运的分子机制的模型，确定跨膜信号转导的机制，并确定转运体-TonB相互作用的调节机制。最后，这些转运蛋白的结构和动力学(基于2桶)同时受到溶质和脂质环境的影响。由于迫切需要建立和解释膜蛋白的高分辨率结构模型，拟议的工作还将量化溶质和脂环境对这类膜蛋白结构的影响。与公共健康相关：拟议的研究将确定细菌通过细胞膜运输稀缺营养的分子机制。这种运输对细菌的生存至关重要，对许多细菌病原体的成功也是至关重要的，例如引起脑膜炎、霍乱和百日咳的细菌。对这些转运机制的了解将有助于开发能够抑制细菌生长的新抗生素。

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 address key questions regarding the 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 inner-membrane protein TonB. These TonB-dependent transporters 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. TonB-dependent transporters are abundant in Gram negative bacterial and are critical to the success of many bacterial pathogens, such as the bacteria that result in meningitis, cholera and pertussis. Because they are unique to bacteria, these transporters are a logical target for the development of new classes of antibiotics. High-resolution crystallographic models have been obtained for a number of TonB-dependent transporters; however, the mechanisms by which transport takes place is unclear. The proposed work will utilize site-directed spin labeling and EPR spectroscopy to test models for the molecular mechanisms of TonB-dependent transport, determine the mechanisms of transmembrane signaling and determine the mechanisms by which the transporter-TonB interaction is regulated. Finally, the structure and dynamics of these transporters (which are based upon 2-barrels) are influenced by both solute and lipid environment. Because of the critical need to generate and interpret high-resolution structural models of membrane proteins, the proposed work will also quantitate the influence of solutes and lipid environment on the structure of this class of membrane proteins. PUBLIC HEALTH RELEVANCE: The proposed research will determine the molecular mechanisms by which bacteria transport scarce nutrients across their cell membrane. This transport is critical to the survival of bacteria, and it is essential for the success of many bacterial pathogens, such as the bacteria that cause meningitis, cholera and pertussis. Knowledge of these transport mechanisms will assist with the development of new antibiotics that can inhibit bacterial growth.