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（申请人提供）：该提案的总体目标是（1）研究膜结合KCNE1蛋白的结构和拓扑；（2）阐明KCNE1与KCNQ1 K+通道C端结构域的结合机制； (3) 确定导致疾病的长 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 KCNE1 蛋白调节 KCNQ1 电压门控 K+ 通道的活性，KCNE1 负责减缓 KCNQ1 (IK) 的电压刺激激活，并且对于正常的通道和心脏功能至关重要。最近发表的 LMPG 胶束中 KCNE1 的溶液 NMR 结构表明，与 LQTS、心房颤动、婴儿猝死综合症和耳聋有关。 KCNE1 采用独特的弯曲 α 螺旋二级结构，多项结构生物学研究表明，当蛋白质嵌入膜中时，胶束中的蛋白质结构会发生巨大变化。与胶束结构相比，二级结构发生了巨大变化 我们认为脂质双层中的 KCNE1 结构与 LMPG 中 KCNE1 的溶液 NMR 结构不同。 KCNQ1 或 KCNE1/KCNQ1 复合物的三维结构尚未确定。此外，E1 与 Q1 结合相互作用的结构性质知之甚少，仅通过生化结合和交联测定进行了间接研究。研究 E1 和 Q1 的结构对于正确描述心跳的功能和节律至关重要，这将有助于直接探测 KCNE1 和 KCNE1 的结构和动态特性。 KCNE1/KCNQ1 复合物将被开发来研究膜中 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该领域的发展。膜蛋白系统（例如 KCNE1）的灵敏度和距离测量 将在具体目标中解决以下相关生物学问题：KCNE1 的哪些片段在双层中是螺旋形的？ KCNE1 有弧形或直形 ¿脂质双层中的螺旋（哪个结构模型是正确的）？ KCNE1 相对于膜的结构拓扑是什么？ KCNE1 如何与 KCNQ1 K+ 通道的细胞质结构域结合？ 哪种结构模型对于 E1 是正确的？ /Q1 复合物？导致疾病的 E1 或 Q1 LQTS 突变会改变 E1/Q1 复合物的结构或结合机制吗？！