Computational Studies of Ion Channels - Resubmission 01

离子通道的计算研究 - 重新提交 01

• 批准号: 9144806
• 负责人: BENOIT ROUX
• 金额: $ 47.19万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9144806
• 关键词: Address; Affect; Arrhythmia; Attention; Bacteria; Behavior; Binding; Biological Models; Cardiac; Cell secretion; Cells; Chemicals; Complex; Computer Assisted; Computer Simulation; Crystallization; Data; Electrophysiology (science); Elements; Engineering; Epilepsy; Family; Free Energy; Goals; Health; Heart; Height; Human; Immune response; Intention; Ion Channel; Ions; Knowledge; Maps; Membrane Proteins; Modeling; Modification; Molecular; Molecular Conformation; Mutation; Nature; Neuraxis; Neurons; Pattern; Physiological; Play; Positioning Attribute; Potassium Channel; Process; Property; Protein Engineering; Recovery; Research; Research Project Grants; Resolution; Role; Site; Structure; Testing; Time; Water; Work; X-Ray Crystallography; base; computer studies; conformational conversion; constriction; innovation; member; molecular targeted therapies; mutant; nervous system disorder; novel; research study; simulation

DESCRIPTION (provided by applicant): The primary goal of this proposal is to define in molecular terms the conformational changes that underlie C- type inactivation in K+ channels, and explain the implications of these conformational changes with regards to ion selectivity. C-type inactivation of K+ channels is a molecular process of great physiological significance and understanding the molecular basis of inactivation has a direct impact on human health. This goal will be achieved using a combination of computational and experimental approaches via 3 specific aims. In Aim 1, we will test the hypothesis that the constricted filter conformation, as observed in several crystal structures of the KcsA channel, is a general property of different K+ channels. Computations are the best way to address this issue to examine three channels that can inactivate and or not inactivate, as well as several mutants of these channels. The impetus to carry this work comes from our recent discovery of the role of inactivating water molecules to explain the origin of the multi-second timescale for recovery in the KcsA channel (Ostmeyer et al, Nature 2013). In Aim 2, we will test the hypothesis that multi-ion effects can explain the difference in ion conduction and selectivity using multi-ion potential of mean force (PMF) computations. In particular, we will test whether the height of the multi-ion free energy barrier through the non-conductive (constricted) state explains the wide range of behaviors in permeation and selectivity that are observed. To buttress our analysis of constricted pores, we will also exploit recent high-resolution data to test the mechanism of selectivity through conductive filters using multi-ion 3d-PMFs. In Aim 3, we will test the hypothetical concept of pore dilation and discover alternative filter conformations using the Tsuka channel as a model system. We recently determined the crystal structure of the Tsuka channel, which displays a novel filter conformation that is wider than usual. Tsuka has about 30% similarity to the NaK channel, which can be converted into a KcsA-like conductive pore with a few mutations. This implies that Tsuka is a relevant member of the K+ channel family. This crystal structure of a novel channel of relevance provides the impetus for this work. In MD simulation, this open dilated pore does not conduct. We will exploit this novel crystal structure to further test the concept of pore dilation and, using MD simulations, examine whether it can behave as a non-conductive inactivated filter. We will also progressively re-engineer the sequence of Tsuka to recapitulate the properties of a K+ selective pore, with the intention of capturing conformational intermediates spanning the range from dilated to conductive selective pore. This sequence/structure transformation will be mapped using X-ray crystallography, electrophysiology, MD simulations, and ROSETTA-DESIGN computer-assisted protein engineering.

描述（由申请人提供）：本提案的主要目标是用分子术语定义 K+ 通道中 C 型失活背后的构象变化，并解释这些构象变化对离子选择性的影响。 K+通道的C型失活是一个具有重要生理意义的分子过程，了解失活的分子基础对人类健康有直接影响。这一目标将通过 3 个具体目标，结合计算和实验方法来实现。在目标 1 中，我们将测试以下假设：在 KcsA 通道的几种晶体结构中观察到的收缩过滤器构象是不同 K+ 通道的一般属性。计算是解决这个问题的最佳方法，以检查可以失活和/或不失活的三个通道，以及这些通道的几个突变体。开展这项工作的动力来自于我们最近发现的水分子失活作用，以解释 KcsA 通道中恢复的多秒时间尺度的起源（Ostmeyer 等人，Nature 2013）。在目标 2 中，我们将测试以下假设：多离子效应可以使用多离子平均力势 (PMF) 计算来解释离子传导和选择性的差异。特别是，我们将测试通过非导电（收缩）状态的多离子自由能垒的高度是否可以解释观察到的渗透和选择性的广泛行为。为了支持我们对收缩孔隙的分析，我们还将利用最新的高分辨率数据来测试使用多离子 3d-PMF 的导电过滤器的选择性机制。在目标 3 中，我们将测试孔隙扩张的假设概念，并使用 Tsuka 通道作为模型系统发现替代的过滤器构象。我们最近确定了 Tsuka 通道的晶体结构，它显示出一种比平常更宽的新型过滤器构象。 Tsuka 与 NaK 通道有约 30% 的相似性，经过少量突变即可转化为类似 KcsA 的导电孔。这意味着 Tsuka 是 K+ 频道家族的相关成员。这种新颖相关通道的晶体结构为这项工作提供了动力。在MD模拟中，这种开放扩张的孔不传导。我们将利用这种新颖的晶体结构来进一步测试孔隙扩张的概念，并使用 MD 模拟来检查它是否可以充当非导电失活过滤器。我们还将逐步重新设计 Tsuka 的序列，以概括 K+ 选择性孔的特性，目的是捕获从扩张选择性孔到导电选择性孔的构象中间体。这种序列/结构转变将使用 X 射线晶体学、电生理学、MD 模拟和 ROSETTA-DESIGN 计算机辅助蛋白质工程来绘制。