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）：该提案的主要目标是用分子术语定义c-型失活的构象变化，并解释这些构型变化在离子选择性方面的含义。 K+通道的C型灭活是一种具有很大生理意义的分子过程，并且了解失活的分子基础对人类健康有直接的影响。通过通过3个特定目的的计算方法和实验方法的组合来实现此目标。在AIM 1中，我们将检验以下假设：在KCSA通道的几个晶体结构中观察到的限制过滤器构象是不同K+通道的一般特性。计算是解决此问题的最佳方法，以检查三个可能失活和或不活化的通道以及这些渠道的几个突变体。携带这项工作的动力来自我们最近发现的灭活水分子的作用来解释KCSA通道中多秒恢复的起源（Ostmeyer等人，自然，2013年）。在AIM 2中，我们将检验以下假设：多离子效应可以使用平均力（PMF）计算的多离子电位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（狭窄）状态的多离子自由能屏障的高度是否解释了观察到的渗透和选择性的广泛行为。为了支撑我们对狭窄毛孔的分析，我们还将利用最近的高分辨率数据来通过使用Multi-ION 3D-PMF来测试选择性的机理。在AIM 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滤波器构象。我们最近确定了Tsuka通道的晶体结构，该结构显示出比平常更宽的新型滤光片构型。 Tsuka与NAK通道具有约30％的相似性，NAK通道可以转换为具有一些突变的KCSA样导电孔。这意味着Tsuka是K+渠道家族的相关成员。新型相关渠道的这种晶体结构为这项工作提供了动力。在MD模拟中，这种开放的扩张孔不会进行。我们将利用这种新型的晶体结构，以进一步测试孔扩张的概念，并使用MD模拟检查它是否可以作为非导电的灭活过滤器。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，以捕获从扩张到导电性选择性孔的范围的构象中间体。该序列/结构转换将使用X射线晶体学，电生理学，MD模拟和Rosetta设计计算机辅助的蛋白质工程绘制。