Structures of Eukaryotic K Channels and Mistic Action

描述(由申请人提供)： 这是一项新的R01提案，题为“真核细胞K通道的结构和Mistic作用”，将集中于两个主要目标：1)Mistic如何工作以允许真核整体膜(IM)蛋白插入和局部发生到大肠杆菌膜中；2)用X射线结晶学方法研究真核细胞电压门控K(Kv)通道的三维结构。我们提出的研究旨在通过关注6个跨膜(TM)-螺旋电压门控K通道的分子作用，将范围和方向扩展到一个新的水平。 在第一个目标中，我们建议研究Mistic的作用机制，Mistic是一种新的伴侣蛋白，它允许真核IM蛋白自主插入到大肠杆菌膜中。我们将分析蛋白质与模型货物蛋白KvPae的相互作用，并与Mistic相关联。这项工作将解决IM蛋白膜拓扑发生的结构基础及其构象灵活性。 在第二个目标中，我们建议确定真核细胞Kv通道的晶体结构。我们将建立一种基于FLAG的抗体与FLAG工程的Kv通道蛋白相结合，以促进它们的结晶。此外，我们将分析真核细胞Kv通道的整个N端结构域Kv1.1与其跨膜结构域之间的蛋白质界面。我们将通过核磁共振波谱分析构象的变化，以阐明通道失活机制的结构基础，包括通道自身的失活亚域和蛋白质与蛋白质的相互作用。 综上所述，我们将主要通过结合X射线结晶学和核磁共振波谱的方法来探索在完整的、分离的真核细胞Kv通道中发生的结构变化。我们将学习大量关于真核细胞Kv通道电压激活和失活的结构机制。

DESCRIPTION (provided by applicant): This is a new R01 proposal entitled "Structures of Eukaryotic K Channels and Mistic Action" will focus on two main goals: 1) understanding how Mistic works to permit insertion and topogenesis of eukaryotic Integral Membrane (IM) proteins into E. coli membrane, and 2) three dimensional structures of eukaryotic voltage-gated K (Kv) channels by x-ray crystallography methods. Our proposed study will aim to expand scope and direction to a newer level by focusing on molecular working of 6 transmembrane (TM)-helix voltage-gated K channels. In the first Aim, we propose to study action mechanisms of Mistic, a novel chaperone protein that permits autonomous insertion of eukaryotic IM proteins into E coli membrane. We will analyze protein interaction with a model cargo protein, KvPae in association with Mistic. This work will address the structural basis of membrane topogenesis of IM proteins and their conformational flexibility. In the second Aim, we propose to determine crystal structure of eukaryotic Kv channels. We will establish a FLAG-based antibody in combination with FLAG-engineered Kv channel proteins to facilitate their crystallization. Furthermore, we will analyze protein interface between the entire N-terminal domain of eukaryotic Kv channel, a Kv1.1 , and its transmembrane domain. We will analyze the conformational change by NMR spectroscopy to address the structural basis of mechanism of channel inactivation by its own inactivation subdomain and by protein-protein interaction. In summary, we will probe structural changes occurring in the intact isolated eukaryotic Kv channels primarily by a combination of x-ray crystallography and NMR spectroscopy methods. We will learn a great deal about structural mechanisms underlying voltage activation and inactivation of eukaryotic Kv channels.