Site-directed spin labeling of ArnT

ArnT 的定点自旋标记

• 批准号: 6711998
• 负责人: CANDICE S KLUG
• 金额: $ 24.6万
• 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/6711998
• 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因素。在感染早期阶段起关键作用的对抗菌肽的细菌耐药性尤其重要。蛋白质和底物涉及细菌（例如沙门氏菌伤寒沙门氏菌和大埃氏菌）发展抗菌肽的抗性的能力。最近鉴定出的参与多层毒素耐药性的蛋白质是一种被称为ARNT的内膜蛋白的基因产物，该蛋白质是将氨基氨基酰胺糖部分转移到脂质A上的，它赋予细菌耐药性向阳离子抗菌抗菌肽多糖苷的抗性。对ARNT中的结构功能关系有更透彻的理解将是制定克服对多粘素和其他阳离子肽的抗性的策略的关键。先前对ARNT的研究都参与了体内酶活性和遗传分析，以确定其在多粘毒素耐药性中的作用。以前尚未通过任何方法对ARNT蛋白进行纯化和研究。该提案的目的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，以便提供有关此新鉴定的转移酶的第一个结构信息。提出了一个模型，其中鼠伤寒沙门氏菌的转移酶由十二个跨膜组成（-Helices;螺旋；该模型将成为SDSL EPR光谱副本对新型蛋白ARNT进行结构评估的基础，然后通过逐步识别Arnt and Indore and Ar a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt and a arnt a arnt a arnt a arnt a arnt a arnt a arnt a arnt a and a and a and a and a and a and进行了启用。使用SDSL EPR光谱法解决：1）创建并表征ARNT的无反应性半胱氨酸构建体； 2）评估该模型预测ARNT由通过假定的跨膜螺旋区域扫描氮氧化物扫描由十二个跨膜α-螺旋组成； 3）通过分析放置在推定的跨膜，表面环和底物结合区域内的一组突变来探索ARNT的整体结构布置； 4）监测底物结合引起的局部和全球结构变化。可以预料，这些研究将为ARNT的局部和全局结构提供见解，ARNT是一种先前未表征的整体膜蛋白，这对于进一步了解我们对膜蛋白的结构和功能动力学的理解至关重要。