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 洛根实验室研究概况和五年目标 (综述)：目前，我们对膜蛋白的结构信息有限。洛根实验室 有兴趣开发新的生物物理方法来探索其结构和动力学性质 应用最新的脉冲EPR波谱技术和膜技术的整合膜蛋白 增溶聚合物。总体目标是研究脂质双层中具有EPR的膜蛋白。 与胶束或洗涤剂相反，因为它更接近于细胞膜。几种蛋白质 与脂质双层相比，已被证明不能在胶束中起作用或正确折叠。这 是具有挑战性的，因为更难表达、纯化和进行生物物理光谱 与胶束或球状体系相比，膜蛋白的实验。我的专业知识是 膜蛋白EPR和样品制备与强大的脉冲EPR仪器联用 (鹿和ESEEM)在我的实验室里，可以测量远距离的东西吸引了几个重要的 有重大生物问题的合作者。我的研究实验室直接与几个 研究人员大幅提高膜蛋白样品制备质量以获得高产量 高质量的鹿数据，导致更准确的结构信息。请参阅支持信。 该实验室的主要生物学重点是直接相关的膜蛋白通道。 心脏疾病。KCNQ1(Q1)是一种具有生物学意义的电压门控钾通道，发现于 受膜蛋白KCNE1(E1)调节的心脏。KCNQ1/KCNE1相互作用缓慢 下调正常通道和心脏功能所需的KCNQ1的激活动力学。遗传性 Q1/E1突变可导致长QT综合征、房颤、婴儿猝死综合征、 心律失常和先天性耳聋。Q1是一种具有六个跨膜结构的膜蛋白。 (TMD)螺旋，前四个TMD形成电压传感器结构域Q1-VSD(S1-S4)，连接到孔 结构域(S5-S6)由S4-S5接头以及胞质N和C-末端结构域组成。三个人- KCNQ1或E1/Q1复合体的空间结构尚未确定。此外， 1与Q1的结合作用/机制的结构性质尚不清楚，只有 用生化结合和交联法进行了间接研究。我们目前正在 应用最先进的EPR技术直接探测Q1的结构和动力学性质 和E1/Q1复合波。 将回答以下相关的生物学问题：KCNQ1的哪些片段是 在脂质双层中是螺旋的？KCNQ1的结构和拓扑结构与 薄膜？Q1如何与功能所需的E1蛋白结合并相互作用？ (实验室5年目标)：(1)开发新的生物物理技术来研究结构和动力学 (2)研究KCNQ1钾离子通道的结构和拓扑结构； 阐明KCNQ1与KCNE1的结合机制；(4)应用膜蛋白 我们开发的技术来研究几个生物重要积分的结构 膜蛋白(流感四聚体M2蛋白，五聚体配基门控离子通道(pLGIC， TRPM8和PIRT)使用脉冲EPR波谱(鹿和ESEEM)。 将开发变革性的生物物理技术来研究结构和动力学 膜蛋白的性质。这些最先进的脉冲EPR波谱技术将 通过显著提高距离测量的敏感度、精确度 所有的膜蛋白系统。此外，一种新的基于聚合物的膜模拟系统将被 这将使研究人员能够更轻松地进行结构和功能测量 在脂质双层中的膜蛋白。聚合物可以微调脂盘的大小以 与膜蛋白复合体的大小相匹配。