Biophysical, Structural and Functional Analysis of Mechanosensitive Channels

机械敏感通道的生物物理、结构和功能分析

• 批准号: 7922636
• 负责人: DOUGLAS CHARLES REES
• 金额: $ 37.22万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7922636
• 关键词: Address; Advisory Committees; Animals; Arabidopsis; Bacteria; Biological Assay; Biological Process; Biology; Cell membrane; Cell physiology; Cells; Characteristics; Chemistry; Collaborations; Coupling; Development; Electrophysiology (science); Environment; Equilibrium; Escherichia coli; Family; Force of Gravity; Growth and Development function; Health; Homologous Gene; Ion Channel; Laboratories; Lead; Lipid Bilayers; Liquid substance; Measurement; Measures; Mechanics; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Morphology; Mutagenesis; Organelles; Organism; Osmotic Pressure; Osmotic Shocks; Perception; Physics; Physiological; Plants; Property; Proteins; Reporter; Research; Role; Series; Stretching; Structure; Surveys; Theoretical model; Thick; Touch sensation; Vesicle; Water; Width; Work; cell growth; improved; interest; protein complex; protein structure; public health relevance; research study; response

DESCRIPTION (provided by applicant): All organisms, from single-celled bacteria to multi-cellular animals and plants, must sense and respond to mechanical force in their external environment (shear force, gravity, touch) and in their internal environment (osmotic pressure, membrane deformation) for proper growth, development, and health. Our research focuses on two families of mechanosensitive channels, the prokaryotic channels MscL and MscS and their eukaryotic homologs in Arabidopsis. MscL and MscS are intrinsically stretch-activated channels that open and close in response to tension applied directly to the bilayer and consequently are sensitive reporters of protein- membrane energetics. Elucidating how these mechanosensitive channels function in the context of the membrane will help us understand how mechanical force can generate biophysical alterations that in turn lead to adaptive changes in cell physiology. Aim 1: Investigate the crystal structures of MscL and MscS in multiple conformational states. MscL and MscS are among the few gated channels that have been crystallographically determined. Our highest priority is to improve the structures of the E. coli channels, but we will also systematically survey prokaryotic homologs and the use of molecular doorstops to trap channels in alternate conformational states to define the gating transition in structural detail. Aim 2: Analyze the biophysical interactions between mechanosensitive channels and the lipid bilayer. Working within the context of a theoretical model, the coupling between gating tension, bilayer thickness, and width of the hydrophobic region of MscL will be explored through mutagenesis and single channel electrophysiology. These studies will dissect the energetic contributions of different membrane deformation terms to the conformational equilibrium between channel states. The physiologically crucial permeation of water through MscL and MscS in giant unilamellar vesicles will be measured volumetrically and compared to the fluid transport properties anticipated from conductance measurements. Aim 3: Characterize functional and structural aspects of eukaryotic MscS-Like channels. The MscS-Like (MSL) channels of Arabidopsis provide an opportunity to investigate the structure and function of mechanosensitive channels in the context of multi-cellular eukaryotic organisms. The oligomeric state, channel characteristics, and structure of these proteins will be investigated. We will also use a series of new and established assays to characterize their biological function in osmotic shock protection, intramembrane localization, electrophysiology and organelle morphology control. Our proposed experiments on the MSLs, together with the experiments proposed above for MscL and MscS, are the start towards a systematic approach to revealing how MS channels function in the context of the membrane and the cell. PUBLIC HEALTH RELEVANCE Force-sensing is a critical aspect of healthy cell growth, morphology and development. We will study in molecular detail how force-sensing is achieved by two families of stretch-activated membrane channels.

描述（由申请人提供）：所有生物体，从单细胞细菌到多细胞动物和植物，都必须感知并响应其外部环境（剪切力、重力、触摸）和内部环境（渗透压、膜变形）中的机械力，以实现正常生长、发育和健康。我们的研究主要集中在两个家庭的机械敏感通道，原核通道MscL和MscS及其真核同源物在拟南芥。MscL和MscS是固有的拉伸激活通道，其响应于直接施加到双层的张力而打开和关闭，因此是蛋白质-膜能量学的敏感报告物。阐明这些机械敏感通道在膜中的功能将有助于我们理解机械力如何产生生物物理改变，从而导致细胞生理学的适应性变化。目的1：研究MscL和MscS在多种构象状态下的晶体结构。MscL和MscS是已经晶体学确定的少数门控通道之一。我们的首要任务是改善E的结构。大肠杆菌通道，但我们也将系统地调查原核同源物和使用分子门挡陷阱通道在交替构象状态，以确定门控转换的结构细节。目的2：分析机械敏感性通道与脂双层之间的生物物理相互作用。在理论模型的背景下工作，门控张力，双层厚度和MscL的疏水区域的宽度之间的耦合将通过诱变和单通道电生理学探索。这些研究将剖析不同的膜变形项的通道状态之间的构象平衡的能量贡献。将体积测量水通过巨单层囊泡中的MscL和MscS的生理关键渗透，并与电导测量预期的流体传输特性进行比较。目的3：表征真核细胞MscS样通道的功能和结构。拟南芥中的MscS-Like（MSL）通道为在多细胞真核生物中研究机械敏感通道的结构和功能提供了机会。这些蛋白质的寡聚状态，通道特性和结构将被研究。我们还将使用一系列新的和已建立的测定来表征它们在渗透休克保护、膜内定位、电生理和细胞器形态控制中的生物学功能。我们提出的MSL实验，以及上面提出的MscL和MscS实验，是揭示MS通道如何在膜和细胞中起作用的系统方法的开始。力感测是健康细胞生长、形态和发育的关键方面。我们将详细研究分子如何通过两个家族的拉伸激活膜通道实现力传感。