Site-directed spin labeling of ArnT

ArnT 的定点自旋标记

• 批准号: 6868210
• 负责人: CANDICE S KLUG
• 金额: $ 22.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-08 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6868210
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; Salmonella typhimurium; antibiotics; arabinose; bacteria infection mechanism; binding sites; conformation; drug resistance; electron spin resonance spectroscopy; enzyme activity; enzyme model; enzyme substrate; membrane proteins; protein structure function; site directed mutagenesis; transferase; virulence

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.

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。细菌对抗菌肽的耐药性在感染早期发挥着关键作用，这一点尤其重要。最近，人们开始根据遗传分析来鉴定与鼠伤寒沙门氏菌和大肠杆菌等细菌对抗菌肽产生耐药性的能力有关的蛋白质和底物。最近发现的参与多粘菌素耐药性的蛋白质是一种内膜蛋白的基因产物，称为 ArnT，它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菌肽多粘菌素的耐药性。更全面地了解 ArnT 的结构-功能关系将是制定克服多粘菌素和其他阳离子肽耐药性策略的关键。先前对 ArnT 的研究均涉及体内酶活性和遗传分析，以确定其在多粘菌素耐药性中的作用； ArnT 蛋白之前尚未通过任何方法进行纯化和研究。该提案的目标是通过定点自旋标记 (SDSL) EPR 光谱研究纯化的内膜蛋白 ArnT 的结构，以便提供有关这种新鉴定的转移酶的第一个结构信息。提出了一种模型，其中鼠伤寒沙门氏菌 ArnT 转移酶由 12 个跨膜（-螺旋）组成；该模型将成为通过 SDSL EPR 光谱法评估新型蛋白质 ArnT 结构的基础，随后检查 ArnT 由于以下原因而发生的结构变化：为了开始提供 ArnT（一种独特的新型膜蛋白）的第一个结构信息，将使用 SDSL EPR 光谱解决以下几点：1）创建并表征ArnT 的无反应性半胱氨酸构建体； 2) 通过硝基氧扫描假定的跨膜螺旋区域来评估预测 ArnT 由十二个跨膜 α 螺旋组成的模型； 3) 通过分析假定的跨膜、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； 4) 监测底物结合引起的局部和整体结构变化。预计这些研究将有助于深入了解 ArnT（一种以前未表征的完整膜蛋白）的局部和整体结构，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。