Spin labeling of MsbA

描述(申请人提供)：被称为三磷酸腺苷结合盒(ABC)转运蛋白的一类蛋白质是自然界中发现的最大的蛋白质之一。它们利用来自三磷酸腺苷的能量在膜双层上移动各种配体的能力或缺乏能力，对细菌生理学和一系列人类病理都是至关重要的。ABC转运蛋白调节多种溶质的进出口，包括抗生素、脂类、化疗药物、糖、氨基酸、盐和金属。 MSBA是脂类A的ABC转运蛋白，存在于革兰氏阴性菌的内膜中，如大肠杆菌。在没有MSBA存在的情况下，细菌细胞在其内膜内积累了有毒数量的类脂A，这是细胞外表面的基本成分。最近获得了MSBA的晶体结构，这为结构和功能动力学研究提供了一个很好的起点。虽然现在已经有了MSBA的结构，但关于它的运输机制仍然存在许多问题。这项提议的目的是利用定点自旋标记(SDSL)电子顺磁共振(EPR)技术，阐明细菌ABC转运蛋白MSBA在其核苷酸结合域与ATP结合以及在其螺旋核心识别和转运脂类底物时发生的构象动力学。 为了解释MSBA同源二聚体在ATP和脂质结合上经历了重要的构象重排，这对其作为脂质输出器的功能是必不可少的，将涉及以下几点：1)评估重组成脂膜的MSBA的四级结构；2)研究ATP结合时MSBA二聚体的构象动力学；以及3)研究脂质结合时MSBA二聚体的构象动力学。 预计这些研究将对MSBA的局部和全球结构动力学产生有价值的见解，因为它发挥着脂质转运体的作用。

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.