EPR Spectroscopic Studies of Membrane Proteins

膜蛋白的 EPR 光谱研究

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要 Lorigan 实验室研究概述和 5 年目标（概述）：目前，我们对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结构和动态特性使用最先进的脉冲 EPR 光谱技术和膜分析整合膜蛋白增溶聚合物。总体目标是研究脂双层中 EPR 的膜蛋白：与胶束或洗涤剂相反，因为它更接近地模仿细胞膜。几种蛋白质与脂质双层相比，已被证明在胶束中无法正常发挥作用或折叠。这具有挑战性，因为表达、纯化和进行生物物理光谱更加困难与胶束或球状系统相比的膜蛋白实验。我的专业知识膜蛋白 EPR 和样品制备以及强大的脉冲 EPR 仪器（DEER 和 ESEEM）在我的实验室中可以测量长距离距离，吸引了几个重要的具有重要生物学问题的合作者。我的研究实验室直接与几个合作研究人员大幅提高膜蛋白样品制备的质量以提高产量高质量的 DEER 数据可带来更准确的结构信息。请参阅支持信。实验室的主要生物学重点是与膜蛋白通道直接相关的膜蛋白通道。心脏病。 KCNQ1 (Q1) 是一种具有生物学意义的电压门控钾通道，存在于心脏受膜蛋白 KCNE1 (E1) 调节。 KCNQ1/KCNE1 相互作用缓慢降低正常通道和心脏功能所需的 KCNQ1 激活动力学。遗传 Q1/E1 突变可导致长 QT 综合征、心房颤动、婴儿猝死综合征、心律失常和先天性耳聋。 Q1是一种具有六次跨膜的膜蛋白 (TMD) 螺旋，前四个 TMD 形成电压传感器域 Q1-VSD (S1-S4)，与孔相连结构域 (S5-S6) 由 S4-S5 接头以及胞质 N 和 C 末端结构域组成。三个- KCNQ1 或 E1/Q1 复合物的维结构尚未确定。此外， E1 与 Q1 的结合相互作用/机制的结构性质知之甚少，并且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 复合体。将回答以下相关生物学问题： KCNQ1 的哪些片段是脂质双层中的螺旋？ KCNQ1 的结构和拓扑是怎样的？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的结合机制； (4)应用膜蛋白我们开发的技术用于研究几个生物学上重要的积分的结构膜蛋白（流感四聚体 M2 蛋白、五聚体配体门控离子通道 (pLGIC、 TRPM8 和 PIRT）使用脉冲 EPR 光谱（DEER 和 ESEEM）。将开发变革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 光谱技术将通过大幅提高距离测量的灵敏度和准确性，推动该领域向前发展所有膜蛋白系统。此外，一种新的基于聚合物的膜模拟系统将开发将使研究人员能够更轻松地进行结构和功能测量脂质双层中的膜蛋白。脂质体的大小可以通过聚合物进行微调与膜蛋白复合物的大小相匹配。