Spin labeling of MsbA

描述（由申请人提供）：一类称为ATP结合盒（ABC）转运蛋白的蛋白质是自然界中发现的最大的蛋白质之一。它们使用来自ATP的能量将各种配体移动穿过膜双层的能力或缺乏这种能力对于细菌生理学和一系列人类病理学至关重要。ABC转运蛋白介导多种溶质的输入和输出，包括抗生素、脂质、化疗剂、糖、氨基酸、盐和金属。 MsbA是脂质A的ABC转运蛋白，存在于革兰氏阴性菌（如大肠杆菌）的内膜中。没有MsbA存在，细菌细胞在其内膜内积累毒性量的脂质A，脂质A是细胞外表面的重要组分。最近获得的MsbA的晶体结构，提供了一个很好的起点，结构和功能动力学研究。虽然MsbA的结构现在是可用的，但关于其运输机制仍存在许多问题。本提案的目的是阐明发生在MsbA，细菌ABC转运蛋白，在其核苷酸结合域结合ATP后，在其螺旋核心的脂质底物的识别和运输，利用定点自旋标记（SDSL）电子顺磁共振（EPR）光谱技术的构象动力学。 为了解决MsbA同源二聚体在ATP和脂质结合时经历对其作为脂质输出体的功能至关重要的显著构象重排的提议，将解决以下几点：1）评价重构到脂质膜中的MsbA的四级结构; 2）研究MsbA二聚体在ATP结合时的构象动力学;和3）研究MsbA二聚体在脂质结合时的构象动力学。 预计这些研究将产生有价值的见解的本地和全球的MsbA的结构动力学，因为它在其作为脂质转运蛋白的作用。

DESCRIPTION (provided by applicant): The class of proteins termed ATP-binding cassette (ABC) transporters is one of the largest found in nature. Their ability, or lack thereof, to move a variety of ligands across a membrane bilayer using energy from ATP is fundamentally important to bacterial physiology and an array of human pathologies. ABC transporters mediate both the import and export of a wide variety of solutes including antibiotics, lipids, chemotherapy agents, sugars, amino acids, salts and metals. MsbA is the ABC transporter for lipid A that is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A, which is an essential component of the outer surface of the cell, within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for structural and functional dynamics studies. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. The goal of this proposal is to elucidate the conformational dynamics that occur in MsbA, a bacterial ABC transporter, upon binding ATP in its nucleotide binding domain and upon recognition and transport of lipid substrates in its helical core, utilizing site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy techniques. In order to address the proposal that the MsbA homodimer undergoes significant conformational rearrangements upon ATP and lipid binding that are essential to its function as a lipid exporter, the following points will be addressed: 1) evaluate the quaternary structure of MsbA reconstituted into lipid membranes; 2) investigate the conformational dynamics of the MsbA dimer upon ATP binding; and 3) investigate the conformational dynamics of the MsbA dimer upon lipid binding. It is anticipated that these studies will produce valuable insights into the local and global structural dynamics of MsbA as it functions in its role as a lipid transporter.