Site-directed spin labeling of ArnT

描述(由申请人提供)：细菌抵抗宿主防御机制的能力是细菌感染毒力的主要贡献因素。细菌对抗菌肽的耐药性尤其显著，抗菌肽在感染的早期阶段起关键作用。最近，基于遗传分析，已经开始鉴定与诸如鼠伤寒沙门氏菌和大肠杆菌等细菌对抗菌肽产生耐药性有关的蛋白质和底物。最近发现的与多粘菌素抗性有关的蛋白质是一种名为Arnt的内膜蛋白的基因产物，它负责将氨基阿拉伯糖部分转移到脂质A上，从而导致细菌对阳离子抗菌肽多粘菌素的抗性。更透彻地了解ARNT的结构-功能关系将是开发克服对多粘菌素和其他阳离子多肽耐药性的策略的关键。以前对Arnt的研究都涉及体内的酶活性和遗传分析，以确定其在多粘菌素抗性中的作用；Arnt蛋白以前没有通过任何方法进行纯化和研究。本研究的目的是利用定点自旋标记(SDSL)EPR波谱技术研究纯化的内膜蛋白ArnT的结构，以提供有关这一新发现的转移酶的第一个结构信息。提出了一个鼠伤寒沙门氏菌Arnt转移酶由12个跨膜螺旋组成的模型，该模型将成为利用SDSL EPR波谱对新蛋白Arnt进行结构评估的基础，并研究由于底物识别而导致的Arnt结构变化。为了开始提供关于Arnt这一独特的新的膜蛋白的第一结构信息，将使用SDSL EPR波谱技术解决以下几点：1)创建并表征Arnt的无反应半胱氨酸结构；2)通过氮氧化物扫描推测的跨膜螺旋区域对预测Arnt由12个跨膜α-螺旋组成的模型进行评估；3)通过分析位于可能的跨膜、表面环和底物结合区的小集合突变来探索Arnt的整体结构安排；以及4)监测底物结合引起的局部和整体结构变化。预计这些研究将为我们深入了解Arnt的局部和全局结构提供帮助，Arnt是一种以前未被描述的完整膜蛋白，这对于进一步了解膜蛋白的结构和功能动力学具有重要意义。

DESCRIPTION (provided by applicant): The ability of bacteria to resist host defense mechanisms is a major contributor to the virulence of bacterial infections. Bacterial resistance to antimicrobial peptides that play a key role in early stages of infection is especially significant. The proteins and substrates involved in the ability of bacteria such as Salmonella typhimurium and Escherichia coil to develop resistance to antimicrobial peptides have recently begun to be identified based on genetic analysis. The most recently identified protein involved in polymyxin resistance is the gene product for an inner membrane protein, termed ArnT, which is responsible for transferring an aminoarabinose moiety onto lipid A, conferring upon the bacteria resistance to the cationic antimicrobial peptide polymyxin. Obtaining a more thorough understanding of structure-function relationships in ArnT will be key to developing strategies to overcome resistance to polymyxin and other cationic peptides. Previous studies of ArnT have all involved in vivo enzymatic activity and genetic analyses to determine its role in polymyxin resistance; the ArnT protein has not previously been purified and studied by any methodology. The goal of this proposal is to study the structure of the purified inner membrane protein ArnT by site-directed spin labeling (SDSL) EPR spectroscopy in order to provide the first structural information on this newly identified transferase. A model is proposed in which the Salmonella typhimurium ArnT transferase is comprised of twelve transmembrane (-helices; this model will become the basis for the structural evaluation of the novel protein ArnT by SDSL EPR spectroscopy followed by the examination of structural changes in ArnT due to substrate recognition. In order to begin providing the first structural information on ArnT, a unique and new membrane protein, the following points will be addressed using SDSL EPR spectroscopy: 1) create and characterize a reactive-cysteine-free construct of ArnT; 2) evaluate the model predicting that ArnT is comprised of twelve transmembrane alpha-helices by nitroxide scanning through a putative transmembrane helical region; 3) explore the overall structural arrangement of ArnT by analyzing small sets of mutations placed within putative transmembrane, surface loop, and substrate binding regions; and 4) monitor local and global structural changes induced by substrate binding. It is anticipated that these studies will provide insights into the local and global structure of ArnT, a previously uncharacterized integral membrane protein, which is of fundamental importance in furthering our understanding of the structure and functional dynamics of membrane proteins.