Computational Studies of Ion Channels - Resubmission 01

描述（由申请人提供）：本提案的主要目标是从分子角度定义K+通道中C型失活的构象变化，并解释这些构象变化对离子选择性的影响。K+通道c型失活是一个具有重大生理意义的分子过程，了解失活的分子基础直接影响人体健康。这一目标将通过三个具体目标使用计算和实验方法的结合来实现。在Aim 1中，我们将测试这样一个假设，即在KcsA通道的几种晶体结构中观察到的收缩滤波器构象是不同K+通道的一般特性。计算是解决这个问题的最佳方法，可以检查可以灭活或不灭活的三个通道，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的发现，即水分子的失活作用可以解释KcsA通道中多秒恢复时间尺度的起源（Ostmeyer等人，Nature 2013）。在目标2中，我们将使用多离子平均力势（PMF）计算来检验多离子效应可以解释离子传导和选择性差异的假设。特别是，我们将测试通过非导电（收缩）状态的多离子自由能势垒的高度是否解释了所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的分析，我们还将利用最近的高分辨率数据来测试使用多离子3d-PMFs通过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滤波器构象。Tsuka与NaK通道的相似性约为30%，通过少量突变可转化为ksa样导电孔。这表明冢是K+通道家族的一个相关成员。这种晶体结构的新型关联通道为这项工作提供了动力。在MD模拟中，这种张开的扩张孔不存在。我们将利用这种新的晶体结构来进一步测试孔隙膨胀的概念，并使用MD模拟来检查它是否可以作为非导电灭活过滤器。我们还将逐步重新设计Tsuka序列，以重现K+选择性孔的性质，旨在捕获从扩张到导电选择性孔的构象中间体。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-DESIGN计算机辅助蛋白质工程来绘制。

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.