Structures of Eukaryotic K Channels and Mistic Action

描述（由申请人提供）： 这是一个新的R 01提案，题为“真核K通道的结构和Mistic行动”将集中在两个主要目标：1）了解Mistic如何工作，以允许插入和拓扑发生的真核整合膜（IM）蛋白到E。大肠杆菌细胞膜; 2）真核细胞电压门控性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通道的分子工作，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，一种新的伴侣蛋白，允许真核IM蛋白自主插入到大肠杆菌膜的作用机制。我们将分析蛋白质与模型货物蛋白KvPae与Mistic的相互作用。这项工作将解决膜拓扑结构的IM蛋白和它们的构象灵活性的基础。 在第二个目标中，我们提出确定真核细胞Kv通道的晶体结构。我们将建立一个基于FLAG的抗体与FLAG工程Kv通道蛋白的组合，以促进其结晶。此外，我们将分析真核细胞Kv通道的整个N-末端结构域（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光谱分析构象变化，以解决通道失活机制的结构基础，通过其自身的失活子域和蛋白质-蛋白质相互作用。 总之，我们将主要通过X射线晶体学和NMR光谱学方法的组合来探测在完整的分离的真核KV通道中发生的结构变化。我们将学习到大量的关于真核细胞Kv通道的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.