EPR Structural Studies of KCNE1/KCNQ1

描述(申请人提供)：这项建议的总体目标是：(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-末端结构域的结合机制；(3)识别致病长QT综合征(LQTS)E1和Q1突变的结构/结合差异；以及(4)发展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K+通道的活性。KCNE1负责减缓电压刺激的KCNQ1(IKS)的激活，对正常的通道和心脏功能是必不可少的。遗传性的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KCNE1在LMPG胶束中的溶液核磁共振结构表明，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，当蛋白质嵌入膜中时，胶束中蛋白质的结构会发生巨大变化。洛根实验室最近对蛋白脂质体中KCNE1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KCNE1在脂质双层中的结构不同于KCNE1在LMPG胶束中的溶液核磁共振结构。KCNQ1或KCNE1/KCNQ1复合体的三维结构尚未确定。此外，对E1与Q1结合作用的结构性质了解甚少，仅通过生化结合和交联法间接研究。要正确描述心跳的功能和节律，关键是要研究E1和Q1的结构。电子顺磁共振波谱将被用来直接探测KCNE1和KCNE1/KCNQ1复合体的结构和动力学性质。将发展变革性的生物物理技术来研究KCNE1和膜中KCNE1/KCNQ1复合体的结构和动力学性质。这些最先进的脉冲EPR光谱技术将极大地提高膜蛋白系统(如KCNE1)的灵敏度和距离测量，从而推动该领域的发展。以下相关的生物学问题将在具体目标中解决：KCNE1的哪些片段在双层中是螺旋的？KCNE1脂双层中是否有弯曲或直的α-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是如何与KCNQ1 K+通道的细胞质结构域结合的？对于E1/Q1复合体，哪个建议的结构模型是正确的？致病的E1或Q1 LQTS突变是否会改变E1/Q1复合体的结构或结合机制？

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?