EPR Structural Studies of KCNE1/KCNQ1

描述（申请人提供）：本研究的总体目标是：（1）研究膜结合KCNE 1蛋白的结构和拓扑结构;（2）阐明KCNE 1与KCNQ 1 K+通道C端结构域的结合机制;（3）鉴定致病性长QT综合征（LQTS）E1和Q1突变的结构/结合差异，（4）发展新的磁共振技术来研究膜蛋白的结构。膜结合的KCNE 1蛋白调节KCNQ 1电压门控K+通道的活性。KCNE 1负责减缓KCNQ 1（IKs）的电压刺激激活，并且对适当的通道和心脏功能至关重要。遗传性E1/Q1突变与LQTS、房颤、婴儿猝死综合征和耳聋有关。最近发表的KCNE 1在LMPG胶束中的溶液NMR结构表明，KCNE 1采用独特的弯曲α-螺旋二级结构。一些结构生物学研究表明，当蛋白质嵌入膜中时，胶束中蛋白质的结构会发生显着变化。Lorigan实验室最近对蛋白脂质体中KCNE 1的CD数据显示，与胶束结构相比，二级结构发生了显着变化。我们假设，KCNE 1的结构在脂质双层不同的溶液NMR结构的KCNE 1在LMPG胶束。KCNQ 1或KCNE 1/KCNQ 1复合物的三维结构尚未确定。此外，E1与Q1的结合相互作用的结构性质知之甚少，仅用生化结合和交联测定间接研究。研究E1和Q1的结构对正确描述心跳的功能和节律至关重要。EPR光谱将用于直接探测KCNE 1和KCNE 1/KCNQ 1复合物的结构和动力学性质。将开发变革性的生物物理技术来研究KCNE 1和KCNE 1/KCNQ 1复合物在膜中的结构和动力学特性。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（如KCNE 1）的灵敏度和距离测量来推动该领域的发展。以下相关的生物学问题将在具体的目标：哪些部分的KCNE 1是螺旋的双层？KCNE 1在脂双层中有一个弯曲的还是直的α-螺旋（哪种结构模型是正确的）？KCNE 1相对于膜的结构拓扑是什么？KCNE 1如何与KCNQ 1 K+通道的胞质结构域结合？下列哪种结构模型对E1/Q1复合体是正确的？致病性E1或Q1 LQTS突变是否会改变E1/Q1复合物的结构或结合机制？

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 α-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?