Tertiary Structures of Potassium Channel Domains

描述（由申请人提供）：这是一个竞争性的延续提案，以支持题为“钾通道域的三级结构”的项目的三个新目标。这一更新方案通过关注6个跨膜螺旋电压门控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，我们提出确定细菌6 - TM - K通道，KvPae复合物与flag导向的Fab复合物。这项工作将探讨电压介导的通道激活的结构基础及其机制，以及通道中赋予的构象灵活性。在第二个目标中，我们提出完成真核生物Kv通道aKv1.1的整个n端结构域的结构，并结合功能表征分析其构象变化。这项工作将解决通道失活机制的结构基础，通过其自身的失活亚域通过蛋白质-蛋白质相互作用。在第三个目标中，我们提出研究钙结合细胞质KChIP蛋白复合物与真核Kv通道的“整个”n端结构域Kv4.2复合物中发生的结构变化。通过x射线晶体学和核磁共振波谱相结合的方法来探测钙诱导的KChIP/通道复合物的构象变化。本研究将扩大我们对真核生物钾通道多种调控机制的分子认识。我们将学习大量关于电压激活（Aim 1）、其子结构域介导的失活（Aim 2）和钙介导的调节（Aim 3）的结构机制。

DESCRIPTION (provided by applicant): This is a competitive continuation proposal to support three new aims of the project entitled "Tertiary structures of potassium channel domains." This renewal proposal expands the 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 determine a bacterial 6 TM K channel, KvPae in complex with an FLAG-directed Fab complex. This work will address the structural basis of voltage-mediated channel activation and its mechanism, and conformational flexibility endowed in the channel. In the second aim, we propose to complete the structure of the entire N-terminal domain of eukaryotic Kv channel, aKv1.1, and analyze the conformational change by NMR spectroscopy combined with functional characterization. This work will address the structural basis of the mechanism of channel inactivation by its own inactivation subdomain by protein-protein interaction. In the third aim, we propose to study structural changes occurring in the complex of calcium-binding cytoplasmic KChIP protein in complex with the "entire" N-terminal domain of eukaryotic Kv channel, Kv4.2. Calcium-induced conformational change in KChIP/channel complex will be probed by a combination of x-ray crystallography and NMR spectroscopy methods. This study will expand our molecular understanding of diverse regulatory mechanisms of eukaryotic potassium channels. We will learn a great deal about structural mechanisms underlying voltage-activation (Aim 1), inactivation mediated by its subdomain (Aim 2), and calcium-mediated regulation (Aim 3).