EPR Spectroscopic Studies of Membrane Proteins

膜蛋白的 EPR 光谱研究

• 批准号: 10619207
• 负责人: GARY A LORIGAN
• 金额: $ 4.52万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10619207
• 关键词: Arrhythmia; Atrial Fibrillation; Binding; Biochemical; Biological; Biological Assay; Biophysics; C-terminal; Cell membrane; Collaborations; Complex; Coupled; Data; Detergents; Disease; Electron Spin Resonance Spectroscopy; Germ-Line Mutation; Goals; Heart; Heart Diseases; Human papillomavirus 16 E1 protein; Influenza; Integral Membrane Protein; Ion Channel; Ion Channel Gating; Kinetics; Letters; Ligands; Link; Lipid Bilayers; Long QT Syndrome; M2 protein; Malignant Neoplasms; Measurement; Measures; Membrane; Membrane Proteins; Methods; Micelles; N-terminal; Nature; Physiologic pulse; Play; Polymers; Potassium Channel; Preparation; Property; Proteins; Research; Research Personnel; Sampling; Structure; Sudden infant death syndrome; System; Techniques; Voltage-Gated Potassium Channel; Work; base; biophysical techniques; biophysical tools; congenital deafness; crosslink; experimental study; heart function; improved; instrumentation; interest; mimetics; protein complex; sensor; spectroscopic survey; three dimensional structure; voltage

Project Summary/Abstract Overview of Research in the Lorigan Lab and 5 Year Goals (Overview): Currently, we have limited structural information on membrane proteins. The Lorigan lab is interested in developing new biophysical methods to probe the structural and dynamic properties of integral membrane proteins using state-of-the-art pulsed EPR spectroscopic techniques and membrane solubilizing polymers. The overall objective is to study membrane proteins with EPR in a lipid bilayer as opposed to a micelle or detergent because it more closely mimics a cell membrane. Several proteins have been shown to not function or fold up correctly in a micelle when compared to a lipid bilayer. This is challenging because it is more difficult to express, purify, and conduct biophysical spectroscopic experiments on membrane proteins when compared to micelle or globular systems. My expertise in membrane protein EPR and sample preparation coupled with the powerful pulsed EPR instrumentation (DEER and ESEEM) in my lab that can measure long range distances has attracted several significant collaborators with important biological problems. My research lab works directly with several researchers to dramatically improve the quality of membrane protein sample preparation to yield high quality DEER data that leads to more accurate structural information. Please see the letters of support. The major biological focus of the lab is on membrane protein channels that are directly related to heart disease. KCNQ1 (Q1) is a biologically significant voltage gated potassium channel found in the heart that is modulated by the membrane protein KCNE1 (E1). KCNQ1/KCNE1 interactions slow down the activation kinetics of KCNQ1 required for proper channel and heart function. Hereditary mutations in Q1/E1 can cause Long-QT syndrome, atrial fibrillation, sudden infant death syndrome, cardiac arrhythmias, and congenital deafness. Q1 is a membrane protein with six transmembrane (TMD) helices, the first four TMDs form the voltage sensor domain Q1-VSD (S1-S4), linked to the pore domain (S5-S6) by the S4-S5 linker and the cytosolic N and C-terminal domains. The three- dimensional structure of KCNQ1 or the E1/Q1 complex has not been determined. Furthermore, the structural nature of the binding interaction/mechanism of E1 with Q1 is poorly understood and has only been investigated indirectly with biochemical binding and cross-linking assays. We are currently applying state-of-the-art EPR techniques to directly probe the structural and dynamic properties of Q1 and the E1/Q1 complex. The following pertinent biological questions will be answered: Which segments of KCNQ1 are helical in a lipid bilayer? What is the structure and topology of the KCNQ1 with respect to the membrane? How does Q1 bind and interact with the E1 protein that is required for function? (5 Year Goals of the Lab): (1) Develop new biophysical techniques to study the structure and dynamics of membrane proteins; (2) Investigate the structure and topology of the KCNQ1 K+ channel; (3) Elucidate the binding mechanism of KCNQ1 with KCNE1; and (4) Apply the membrane protein techniques that we develop to investigate the structure of several biologically important integral membrane proteins (influenza tetrameric M2 protein, Pentameric ligand-gated ion channels (pLGIC, TRPM8 and PIRT) using pulsed EPR spectroscopy (DEER and ESEEM). Transformative biophysical techniques will be developed to study the structural and dynamic properties of membrane proteins. These state-of-the-art pulsed EPR spectroscopic techniques will move the field forward by dramatically increasing sensitivity, accuracy of distance measurements for all membrane protein systems. Also, a new polymer based membrane mimetic system will be developed that will enable researchers to more easily conduct structural and functional measurements of membrane proteins in a lipid bilayer. The size of the lipodisqs can be fine-tuned by the polymer to match the size of the membrane protein complex.

项目摘要/摘要 洛里根实验室的研究概述和5年目标 （概述）：目前，关于膜蛋白的结构信息有限。洛里根实验室是 有兴趣开发新的生物物理方法来探测 使用最先进的EPR光谱技术和膜的整体膜蛋白 溶解聚合物。总体目的是在脂质双层中使用EPR研究膜蛋白作为 与胶束或洗涤剂相反，因为它更紧密地模拟细胞膜。几种蛋白质 与脂质双层相比，已显示出在胶束中不正常或在胶束中正确折叠。这 具有挑战性，因为它更难表达，纯化和进行生物物理光谱镜 与胶束或球状系统相比，膜蛋白上的实验。我的专业知识 膜蛋白EPR和样品制备与强大的脉冲EPR仪器结合 （鹿和ESEEM）在我的实验室中可以测量远距离距离的人吸引了几个重要的 有重要生物学问题的合作者。我的研究实验室直接与几个 研究人员显着提高膜蛋白样品制剂的质量以产生高 质量鹿数据，可提供更准确的结构信息。请参阅支持信。 实验室的主要生物学重点是直接相关的膜蛋白通道 心脏病。 KCNQ1（Q1）是一种在生物学上具有重要意义的电压门控钾通道 由膜蛋白KCNE1（E1）调节的心脏。 KCNQ1/KCNE1相互作用缓慢 在适当的通道和心脏功能所需的KCNQ1的激活动力学下。遗传 Q1/E1中的突变会引起长QT综合征，房颤，猝死综合征， 心律不齐和先天性耳聋。 Q1是具有六个跨膜的膜蛋白 （TMD）螺旋，前四个TMD形成电压传感器域Q1-VSD（S1-S4），链接到孔 S4-S5接头以及胞质N和C末端域的域（S5-S6）。三 尚未确定KCNQ1或E1/Q1复合物的尺寸结构。此外， E1与Q1的结合相互作用/机制的结构性知之甚少，仅具有 通过生化结合和交联测定间接研究。我们目前 应用最先进的EPR技术直接探测Q1的结构和动态特性 和E1/Q1复合物。 将回答以下相关的生物学问题：KCNQ1的哪些段是 螺旋中的脂质双层？ KCNQ1的结构和拓扑是什么 膜？ Q1如何与功能所需的E1蛋白相互作用？ （实验室的5年目标）：（1）开发新的生物物理技术来研究结构和动态 膜蛋白； （2）研究KCNQ1 K+通道的结构和拓扑； （3） 阐明KCNQ1与KCNE1的结合机理； （4）应用膜蛋白 我们开发的技术来研究几种具有生物学重要积分的结构 膜蛋白（流感四聚体M2蛋白，五聚合配体门控离子通道（PLGIC，PLGIC，， TRPM8和PIRT）使用脉冲EPR光谱（鹿和ESEEM）。 将开发转化性生物物理技术来研究结构和动态 膜蛋白的特性。这些最先进的脉冲EPR光谱技术将 通过显着提高灵敏度，距离测量的准确性来向前移动田地 所有膜蛋白系统。此外，新的基于聚合物的膜模拟系统将是 开发的将使研究人员更容易地进行结构和功能测量 脂质双层中的膜蛋白。聚合物可以微调脂肪曲的大小 匹配膜蛋白复合物的大小。