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者。细菌对在感染的早期阶段起关键作用的抗菌肽的耐药性尤其重要。涉及细菌（例如鼠伤寒沙门氏菌和大肠杆菌）产生抗微生物肽抗性的能力的蛋白质和底物最近已开始基于遗传分析来鉴定。最新鉴定的涉及多粘菌素抗性的蛋白质是内膜蛋白的基因产物，称为ArnT，其负责将氨基阿拉伯糖部分转移到脂质A上，从而赋予细菌对阳离子抗微生物肽多粘菌素的抗性。获得更透彻的了解结构-功能关系的ArnT将是关键的发展战略，以克服耐药性多粘菌素和其他阳离子肽。以前的研究都涉及在体内的酶活性和遗传分析，以确定其在多粘菌素耐药性的作用，ArnT蛋白以前没有被纯化和研究的任何方法。本提案的目的是研究纯化的内膜蛋白ArnT的结构，通过定点自旋标记（SDSL）EPR光谱，以提供第一个结构信息，这个新发现的转移酶。提出了一个模型，其中鼠伤寒沙门氏菌ArnT转移酶是由12个跨膜（-螺旋），该模型将成为基础的结构评价的新蛋白质ArnT的SDSL EPR光谱其次是检查的结构变化，由于底物识别的ArnT。为了开始提供关于ArnT（一种独特的新的膜蛋白）的第一个结构信息，将使用SDSL EPR光谱来解决以下几点：1）创建并表征ArnT的无反应性半胱氨酸的构建体; 2）通过氮氧扫描通过推定的跨膜螺旋区域来评估预测ArnT由12个跨膜α-螺旋组成的模型; 3）通过分析位于假定的跨膜、表面环和底物结合区域内的小组突变来探索ArnT的整体结构排列;以及4）监测由底物结合诱导的局部和全局结构变化。预计这些研究将提供深入了解的本地和全球结构的ArnT，以前未表征的整合膜蛋白，这是根本的重要性，在进一步了解膜蛋白的结构和功能动力学。

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.