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

项目总结/摘要 机械敏感（MS）通道是一种低聚膜蛋白，其功能是机械-电 感官开关在广泛的生理过程。这些包括触觉、听觉、本体感觉、膨压 植物细胞中的控制和细菌中的转录调节。其中，MS通道的基本类别 通过经历主要的结构转变来响应脂质双层物理性质的变化， 对膜张力的反应，从而在生物体对机械张力的反应中发挥主要作用。 刺激。这被称为机械敏感性的“来自脂质的力”原理。 本计画的总体、长期目标是了解“来自脂质的力”的分子机制 机械敏感通道的门控。具体来说，我们将重点介绍MscS系列MS通道， 大多数原核生物和植物。这些通道在各种生理事件中具有根本的重要性， 可以被设计用于生物医学应用，并在细胞膜中显示出迷人的膜内异质性。 直系亲属更重要的是，MscS家族为我们提供了研究功能的可能性， 行为，高分辨率结构和动力学在同一个MS系统。 尽管已经广泛地研究了MscL和MscS通道，并且已经研究了晶体结构。 虽然有多种构象，但仍有一些主要的机理问题有待解决。这是 特别是对于通道门控的分子事件，根据令人兴奋的新的初步数据， 这一提议的核心。在这方面，我们计划通过实验解决几个基本问题： 能量转换步骤的物理基础是什么，从跨双层张力开始， 在蛋白质运动中？在其天然的双层嵌入形式中，关键功能状态的结构是什么？ 机械传导发生在分子的什么地方？怎么做？ 功能研究将旨在了解能量转导的物理基础。信息 关于MSCS的架构，动态和充满活力的关系（加上其他相关成员） 超家族）与其周围的脂质双层将获得从冷冻-EM，EPR分析自旋标记的 突变体和计算方法。数据将被解释以产生高分辨率的结构， 每种通道中门控通路的不同阶段。我们认为新的冷冻电镜的出现 分析膜蛋白在其天然环境中的结构和动力学的方法应 开辟了一条令人兴奋的新实验途径，将有助于理解生物学上重要的 事件，如离子通道门控、伤害感受和信号转导。

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.