EPR Structural Studies of KCNE1/KCNQ1

KCNE1/KCNQ1 的 EPR 结构研究

• 批准号: 8724535
• 负责人: GARY A LORIGAN
• 金额: $ 26.98万
• 依托单位: MIAMI UNIVERSITY OXFORD
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8724535
• 关键词: Address; Adopted; Atrial Fibrillation; Binding; Biochemical; Biological; Biological Assay; C-terminal; Complex; Cytoplasmic Tail; Data; Disease; Electron Spin Resonance Spectroscopy; Goals; Inherited; Integral Membrane Protein; KCNQ1 protein; Link; Lipid Bilayers; Liposomes; Location; Long QT Syndrome; Magnetic Resonance; Measurement; Membrane; Membrane Proteins; Methods; Micelles; Mutation; NIH Program Announcements; Nature; Physiologic pulse; Point Mutation; Potassium Channel; Property; Proteins; Publishing; Research; Sampling; Solutions; Spectrum Analysis; Structural Models; Structure; Sudden infant death syndrome; System; Techniques; United States National Institutes of Health; base; biochemical model; biophysical techniques; crosslink; data modeling; deafness; heart function; improved; mutant; proteoliposomes; public health relevance; simulation; structural biology; three dimensional structure; voltage

DESCRIPTION (provided by applicant): The overall goals of this proposal are (1) investigate the structure and topology of the membrane-bound KCNE1 protein; (2) elucidate the binding mechanism of KCNE1 with the C-terminal domain of the KCNQ1 K+ channel; (3) identify structural/binding differences in disease-causing long QT syndrome (LQTS) E1 and Q1 mutations, and (4) develop new magnetic resonance techniques to study the structure of membrane proteins. The membrane-bound KCNE1 protein modulates the activity of the KCNQ1 voltage-gated K+ channel. KCNE1 is responsible for slowing the voltage-stimulated activation of KCNQ1 (IKs) and is essential for proper channel and heart function. Hereditary E1/Q1 mutations have been linked to LQTS, atrial fibrillation, sudden infant death syndrome, and deafness. A recently published solution NMR structure of KCNE1 in LMPG micelles reveals that KCNE1 adopts a unique curved alpha-helical secondary structure. Several structural biology studies have indicated that the structure of a protein in a micelle can change dramatically when the protein is embedded in a membrane. Recent CD data by the Lorigan lab on KCNE1 in proteoliposomes reveals dramatic changes in the secondary structure when compared to the micelle structure. We hypothesize that the structure of KCNE1 in a lipid bilayer differs from the solution NMR structure of KCNE1 in LMPG micelles. The three-dimensional structure of KCNQ1 or the KCNE1/KCNQ1 complex has not been determined. Furthermore, the structural nature of the binding interaction of E1 with Q1 is poorly understood and has only been investigated indirectly with biochemical binding and cross-linking assays. It is critical to study the structureof E1 with Q1 to properly describe the function and rhythm of a heartbeat. EPR spectroscopy will be used to directly probe the structural and dynamic properties of KCNE1 and the KCNE1/KCNQ1 complex. Transformative biophysical techniques will be developed to study the structural and dynamic properties of KCNE1 and the KCNE1/KCNQ1 complex in a membrane. These state-of-the-art pulsed EPR spectroscopic techniques will move the field forward by dramatically increasing sensitivity and distance measurements of membrane protein systems such as KCNE1. The following pertinent biological questions will be addressed in the specific aims: Which segments of KCNE1 are helical in a bilayer? Does KCNE1 have a curved or straight ¿-helix in a lipid bilayer (which structural model is correct)? What is the structural topology of KCNE1 with respect to the membrane? How does KCNE1 bind to the cytoplasmic domain of the KCNQ1 K+ channel? Which proposed structural model is correct for the E1/Q1 complex? Do disease-causing E1 or Q1 LQTS mutations alter the structure or binding mechanism of the E1/Q1 complex?!

描述（由适用提供）：该提案的总体目标是（1）研究膜结合的KCNE1蛋白的结构和拓扑； （2）阐明KCNE1与KCNQ1 K+通道的C末端结构域的结合机理； （3）确定引起疾病的长QT综合征（LQTS）E1和Q1突变的结构/结合差异，以及（4）开发新的磁共振技术来研究膜蛋白的结构。膜结合的KCNE1蛋白调节KCNQ1电压门控的K+通道的活性。 KCNE1负责减慢KCNQ1（IK）的电压刺激激活，对于适当的通道和心脏功能至关重要。遗传性E1/Q1突变与LQT，心房颤动，猝死综合症和死亡有关。 KCNE1在LMPG胶束中的最近发表的溶液NMR结构表明，KCNE1采用了独特的弯曲α-螺旋二级结构。几项结构生物学研究表明，胶束中蛋白质的结构在将蛋白质嵌入膜中时会发生巨大变化。 Lorigan Lab在蛋白脂质体中KCNE1上的CD数据最近发现与胶束结构相比，二级结构的急剧变化。我们假设在LMPG胶束中KCNE1的溶液NMR结构中，KCNE1的结构在脂质双层中的差异。尚未确定KCNQ1或KCNE1/KCNQ1复合物的三维结构。此外，E1与Q1的结合相互作用的结构性知之甚少，并且仅通过生化结合和交联测定法间接研究。使用Q1研究E1的结构至关重要，以正确描述心跳的功能和节奏。 EPR光谱法将用于直接探测KCNE1和KCNE1/KCNQ1复合物的结构和动态特性。将开发转化性生物物理技术来研究膜中KCNE1和KCNE1/KCNQ1复合物的结构和动态特性。这些最先进的脉冲EPR光谱技术将通过显着提高膜蛋白系统（例如KCNE1）的灵敏度和距离测量值来向前移动该领域。具体目的将解决以下相关生物学问题：KCNE1的哪个段是双层中的？ KCNE1在脂质双层中是否具有弯曲或直helix（哪种结构模型正确）？ KCNE1相对于膜的结构拓扑是什么？ KCNE1如何结合KCNQ1 K+通道的细胞质结构域？哪种建议的结构模型对于E1/Q1复合物是正确的？引起疾病的E1或Q1 LQTS突变会改变E1/Q1复合物的结构或结合机制吗？