Structural Basis of “Force from Lipids” Activation in Mechanosensitive Channels

项目摘要/摘要 机械敏感(MS)通道是一种具有机械电子功能的寡聚膜蛋白 感觉开关在广泛的生理过程中。这些包括触觉、听觉、本体感觉、膨胀 植物细胞的控制和细菌的渗透调节。其中，基本的一类MS通道 通过经历主要的结构转变来响应脂质双层的物理性质的变化 对膜张力的反应，从而在生物对机械压力的反应中发挥重要作用 刺激物。这被称为机械敏感性的“来自脂类的力”原理。 这个项目的总体、长期目标是了解“来自脂类的力量”的分子机制。 机械敏感通道中的门控。具体地说，我们将重点介绍位于 大多数原核生物和植物。这些通道在各种生理事件中具有基本重要性， 可用于生物医学应用，并显示出令人着迷的膜内异质性 家庭同源异物。更重要的是，MSCs家族为我们提供了研究功能性 行为、高分辨率结构和动力学在同一个MS系统中。 尽管对MSCL和MSCs通道进行了广泛的研究，并对其晶体结构 尽管有多种构象可供选择，但仍有一些重大的机制问题有待解决。这是 鉴于令人兴奋的新的初步数据，尤其是对于通道门控背后的分子事件来说 这项提议的核心是。在这方面，我们计划试验性地解决几个基本问题： 能量传递步骤的物理基础是什么，从跨双分子层张力开始到最终 在蛋白质运动中？在其自然的双层嵌入形式中，关键功能状态的结构是什么？ 在分子中的什么地方发生了机械转导？那又是怎么做的呢？ 功能研究将旨在了解能量转导的物理基础。信息 关于MSCS(以及其他相关成员)的架构、动力学和能量关系 超家族)及其周围的脂质双层将从自旋标记的冷冻-EM，EPR分析中获得 突变体和计算方法。数据将被解释为生成高分辨率的 在每种类型的通道中，门控通路的不同阶段。我们认为，新的低温EM的出现 膜蛋白在天然环境中的结构和动力学分析方法应 开辟一条激动人心的新的实验途径，将有助于理解生物学上的重要 离子通道门控、伤害性感受和信号转导等事件。

Project Summary/Abstract Mechanosensitive (MS) channels are oligomeric membrane proteins that function as mechano-electrical sensory switches in a wide range physiological processes. These include touch, hearing, proprioception, turgor control in plant cells and osmoregulation in bacteria. Among these, a fundamental class of MS channels responds to changes in the physical properties of the lipid bilayer by undergoing major structural transitions in response to membrane tension, thus fulfilling a major role in the response of living organisms to mechanical stimuli. This has been referred to as the “force from lipid” principle of mechanosensitivity. The overall, long-term goal of this project is to understand the molecular mechanism of “force from lipid” gating in mechanosensitive channels. Specifically, we will focus on the MscS family of MS channels found in most prokaryotes and plants. These channels are of fundamental importance in various physiological events, can been engineered for biomedical applications, and display fascinating intramembrane heterogeneity among family orthologs. More importantly, the MscS family Affords us the possibility of studying the functional behavior, high resolution structure and dynamics in the same MS system. Although MscL and MscS channels have been studied extensively and crystal structures have been available in multiple conformations, there are still major mechanistic questions that remain to be solved. This is particularly true for the molecular events underlying channel gating, in light of exciting new preliminary data at the core of this proposal. In this respect, we plan to experimentally address several fundamental questions: What is the physical basis of the energy transduction steps, starting with trans-bilayer tension and culminating in protein motion? What are the structures of the key functional states in its native, bilayer-embedded form? Where in the molecule does mechanical transduction occur? And how? Functional studies will be designed to understand the physical basis of energy transduction. Information on the architecture, dynamics and energetic relationship of MscS (plus other related members of the superfamily) with its surrounding lipid bilayer will be obtained from cryo-EM, EPR analysis of spin labeled mutants and computational methods. The data will be interpreted to generate high resolution structures of the different stages of the gating pathway in each type of channel. We suggest that the advent of new cryo-EM approaches to the analysis of structure and dynamics in membrane proteins in their native environment shall open an exciting new experimental avenue that will contribute to the understanding of biologically important events such as ion channel gating, nociception and signal transduction.