Spin Labeling of MsbA

描述(申请人提供)：被鉴定为三磷酸腺苷结合盒(ABC)转运体的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来说都是至关重要的。ABC转运蛋白调节多种溶质的进出口，包括抗生素、化疗药物和脂类。具体地说，MSBA是脂类A的ABC转运蛋白，这种转运蛋白存在于革兰氏阴性细菌的内膜，如大肠杆菌、鼠伤寒沙门氏菌和霍乱弧菌。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。虽然MSBA的晶体结构已经被解决，但原始结构被撤回，重新分析的数据最近被重新发布为新结构。尽管如此，关于其功能结构仍有许多问题。而且，在ABC转运蛋白中，在催化循环中分子水平上的确切作用机制在很大程度上仍然不清楚，MSBA转运脂A的机制还没有建立起来。在上一个资助期，我们的团队已经在MSBA中确定了底物结合关键区域的局部动态，增加了对这一基本蛋白质的详细知识。负责ATP水解和脂类A运输的功能或功能障碍的每个部位的作用是有价值的信息，目前其他方法无法轻易获得。确定许多这些保守的位点在运输过程中发挥作用的具体步骤将极大地提高我们目前对这类重要转运蛋白的功能理解。对完全活跃的转运蛋白和功能失调的转运蛋白的功能的洞察，是我们从根本上理解一整类潜在的药物和基因治疗靶点的关键。因此，将结合生化活性分析和定点自旋标记(SDSL)电子顺磁共振(EPR)技术，使用循序渐进的方法来测试MSBA同源二聚体通过改变二聚体界面的位置和ATP和脂质A结合上的核苷酸结合结构域进行显著的局部和全局构象重排的提议。与公共卫生相关：多药耐药性是医学上的一个严重问题，不仅在传染病的治疗中往往无效，而且在癌症患者的治疗中也是如此。此外，由于MSBA与其他与囊性纤维化跨膜电导调节等常见遗传病有关的ABC转运蛋白非常相似，通过研究易于纯化的MSBA获得的任何新的功能信息都将有助于我们理解这类非常重要的整膜蛋白的结构与功能关系。MSBA与其一般类别中的许多蛋白质的相似性，以及它在病原菌中的流行，为详细研究这种转运蛋白创造了机会，这将增加我们对一整类潜在的新型药物靶点的基础知识。

DESCRIPTION (provided by applicant): The class of proteins identified as 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 to an array of human pathologies. ABC transporters mediate both the import and export of a wide variety of solutes including antibiotics, chemotherapy agents, and lipids. Specifically, MsbA is the ABC transporter for lipid A that is found in the inner membranes of Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, and Vibrio cholerae. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A, which is an essential component of the outer surface of the cells, within their inner membranes. Although crystal structures have been solved for MsbA, the original structures were retracted and the reanalyzed data were recently republished as new structures. Still, many questions remain concerning its functional structure. And, the exact mechanisms of function on the molecular level during the catalytic cycle remain largely unidentified in ABC transporters in general, and the mechanism by which MsbA transports lipid A has yet to be established. The local dynamics of regions key to substrate binding have been determined in MsbA by our group in the last funding period, adding detailed knowledge of this essential protein. The role of each site responsible for the function or dysfunction of ATP hydrolysis and transport of lipid A is valuable information not readily attainable by other methods at this time. Identifying the specific steps in which many of these conserved sites play in the transport process will greatly improve our current functional understanding of this important class of transporters. Insights into the function of both fully active as well as dysfunctional transporters are key to our fundamental understanding of an entire class of potential drug and gene therapy targets. Thus, the proposal that the MsbA homodimer undergoes significant local and global conformational rearrangements through changes in the location of the dimer interface and within the nucleotide binding domains upon ATP and lipid A binding that are essential to its function as a lipid A exporter will be tested using a step by step approach to function using a combination of biochemical activity assays and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy techniques. PUBLIC HEALTH RELEVANCE: Multidrug resistance is a serious problem in medicine, not only in the often futile treatment of infectious diseases, but also in the treatment of cancer patients. In addition, because of the close similarity of MsbA with other ABC transporters implicated in various common genetic disorders such as the cystic fibrosis transmembrane conductance regulator, any new functional information gained by studying the easily purified MsbA will be beneficial to our understanding of structure-function relationships in this very important class of integral membrane proteins. The similarity of MsbA to so many proteins in its general class, and its prevalence in pathogenic bacteria, creates an opportunity for the detailed study of this transporter that will add to our fundamental knowledge of an entire class of potential novel drug targets.