Structural Dynamics of Mechanosensitive Channels

机械敏感通道的结构动力学

• 批准号: 7236367
• 负责人: Eduardo A Perozo
• 金额: $ 14.93万
• 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: 2001-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7236367
• 关键词: Escherichia coli; bioenergetics; biological signal transduction; biomechanics; electron spin resonance spectroscopy; electrophysiology; intermolecular interaction; lipid bilayer membrane; mechanical pressure; mechanical stress; mechanoreceptors; membrane channels; membrane permeability; membrane proteins; membrane structure; protein structure function

DESCRIPTION (provided by applicant): Mechanosensitive (MS) channels are oligomeric membrane proteins that respond to changes in bilayer tension by catalyzing the transfer of ions and other solutes across the membrane, fulfilling a major role in the response of living organisms to mechanical stimuli. These channels are considered to function as mechano-electrical switches in such diverse physiological processes as touch, hearing, proprioception, turgor control in plant cells and osmoregulation in bacteria. The overall, long-term goal of this project is to understand the molecular mechanism of gating in prokaryotic mechanosensitive channels. Although the recent determination of the MscL and MscS crystal structures has dramatically improved our knowledge of this class of molecules, a number of mechanistic questions remain to be solved. This is particularly true for the molecular events underlying channel gating. In this respect, we plan to experimentally address several fundamental questions: What regions of the channel form the gate(s) and how do they move to produce gating? What is the physical basis of the energy transduction steps, starting with transbilayer tension and culminating in protein motion? Where in the molecule does mechanical transduction occur? What are the structures of the key functional states? The fact that MscL and MscS can be activated by pressure gradients both in native membrane and after reconstitution in pure lipid systems indicates that these channels are gated directly by tension transmitted through the bilayer. Therefore, establishing the physical principles underlying the energy transduction steps in these proteins will require studying the role of protein-lipid interactions in this process. The approach we plan to pursue combines reporter-group spectroscopic techniques (spin labeling/EPR, Fluorescence) and electrophysiological methods with classical biochemical and molecular biological procedures. Functional studies will be targeted to understand the physical basis of energy transduction in mechanosensitive channels. Information on the topology, secondary, and tertiary structure of MscS and MscL will be obtained from EPR analysis of spin labeled mutants. The data will be interpreted to generate backbone models of the different stages of the gating pathway in each type of channel. This proposal opens up a new experimental avenue that will contribute to the understanding of biologically important events such as ion channel gating, nociception and signal transduction.

描述（由申请人提供）：机械敏感（MS）通道是寡聚膜蛋白，它们通过催化离子和其他溶质在整个膜中的转移而响应双层张力的变化，从而在生物体对机械刺激的响应中扮演着重要作用。这些通道被认为在诸如触摸，听力，本体感受，植物细胞中的turgor控制等多种生理过程中充当机械 - 电动开关。该项目的总体长期目标是了解原核生物机械敏感通道中门控的分子机制。尽管最近对MSCL和MSC晶体结构的确定已大大提高了我们对这类分子的了解，但仍有许多机械问题待解决。对于基础通道门控的分子事件尤其如此。在这方面，我们计划在实验中解决几个基本问​​题：渠道的哪些区域形成了大门，以及它们如何产生门控？从跨贝张张力开始并在蛋白质运动中达到顶点，能量转导步骤的物理基础是什么？机械转导在哪里发生？关键功能状态的结构是什么？ MSCL和MSC可以被天然膜中的压力梯度和重建后的压力梯度激活，这表明这些通道是通过通过双层传播的张力直接封闭的。因此，建立这些蛋白质中能量转导步骤的基础的物理原理将需要研究蛋白 - 脂质相互作用在此过程中的作用。我们计划采用的方法将记者组光谱技术（自旋标记/EPR，荧光）和电生理方法与经典生化和分子生物学程序结合在一起。功能研究将是针对性的，以了解机械敏感通道中能量转导的物理基础。 MSC和MSCL的拓扑，次级和第三级结构的信息将从自旋标记突变体的EPR分析中获得。数据将被解释为在每种类型的通道中生成门控途径不同阶段的骨干模型。该提案开辟了一个新的实验途径，该途径将有助于理解生物学上重要的事件，例如离子通道门控，伤害感受和信号转导。