EPR Spectroscopic Studies of Membrane Proteins

项目总结/摘要 Lorigan实验室的研究概述和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 感兴趣的是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 增溶聚合物。总体目标是研究具有EPR的脂质双层中的膜蛋白， 与胶束或去污剂相反，因为它更接近于模仿细胞膜。几种蛋白质 与脂双层相比，已显示在胶束中不能正确地起作用或折叠。这 具有挑战性，因为它更难表达、纯化和进行生物物理光谱 与胶束或球状系统相比，对膜蛋白的实验。我的专长是 膜蛋白EPR和样品制备与强大的脉冲EPR仪器相结合 (DEER和ESEEM）在我的实验室，可以测量长距离的距离吸引了几个显着的 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备的质量， 高质量的DEER数据，从而获得更准确的结构信息。请看支持信。 该实验室的主要生物学重点是直接相关的膜蛋白通道 心脏病KCNQ 1（Q1）是一种具有生物学意义的电压门控钾通道，发现于 由膜蛋白KCNE 1（E1）调节的心脏。KCNQ 1/KCNE 1相互作用缓慢 KCNQ 1的激活动力学需要适当的通道和心脏功能。遗传性 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是一种具有六个跨膜区的膜蛋白， (TMD)螺旋，前四个TMD形成连接到孔的电压传感器域Q1-VSD（S1-S4 结构域（S5-S6）通过S4-S5接头和胞质N和C末端结构域连接。三个- KCNQ 1或E1/Q1复合物的三维结构尚未确定。而且 E1与Q1的结合相互作用/机制的结构性质知之甚少， 已经用生物化学结合和交联测定法间接地研究了。我们目前正在 应用最先进的EPR技术直接探测Q1的结构和动力特性 和E1/Q1复合体。 将回答以下相关的生物学问题：KCNQ 1的哪些片段是 脂质双层中的螺旋结构？KCNQ 1相对于 膜？Q1如何与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，研究结构和动力学 （2）研究KCNQ 1钾通道的结构和拓扑结构;（3） 阐明KCNQ 1与KCNE 1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， TRPM 8和PIRT）使用脉冲EPR光谱（DEER和ESEEM）。 将开发变革性的生物物理技术，以研究 膜蛋白的性质。这些最先进的脉冲EPR光谱技术将 通过大幅提高灵敏度、距离测量的准确性， 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容易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。

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.