Enabling physical stimuli in the study of structural dynamics: The sensory ion channels

在结构动力学研究中启用物理刺激：感觉离子通道

• 批准号: 10221608
• 负责人: Simon Scheuring
• 金额: $ 118.65万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221608
• 关键词: 3-Dimensional; Area; Atomic Force Microscopy; Biological; Biomedical Research; Chemicals; Complement; Cryoelectron Microscopy; Crystallization; Cues; Detection; Development; Electrons; Engineering; Exposure to; Fluorescence Resonance Energy Transfer; Image; Ion Channel; Kinetics; Lateral; Lead; Medicine; Membrane; Membrane Proteins; Methodology; Modality; Molecular Conformation; Molecular and Cellular Biology; Pathology; Physiological; Piezo ion channels; Proteins; Resolution; Retinal blind spot; Roentgen Rays; Sensory; Speed; Stimulus; Structure; TRPV channel; Techniques; Technology; Temperature; Text; Time; Voltage-Gated Potassium Channel; X ray diffraction analysis; algorithm development; classification algorithm; direct application; improved; insight; instrumentation; movie; new technology; novel; particle; physical process; protein structure; response; structural biology; temporal measurement; tool; voltage

Simon Scheuring, Weill Cornell Medicine Scientific Area : 6 MCB : Molecular and Cellular Biology / 5 IE : Instrumentation and Engineering Project Summary/Abstract (30 lines of text): In the recent years, we have seen tremendous progress in structural biology owing to breakthroughs in 3D- crystallization methodology for X-ray diffraction and improved particle classification algorithms and the development of direct electron detection for cryo-EM. As a result, membrane protein structure resolution is now rather routine and progresses at a pace of almost 2 structures per week. To complement structures, technologies like FRET, EPR and HDX give invaluable insights into the range of dynamics and kinetics of conformational states. All experimental structural and dynamical techniques have however a blind spot: they are poorly adapted to analyze proteins in response to physical stimuli such as force, temperature and voltage. This is particularly regrettable for the case of sensory ion channels that process these physical stimuli, because they are involved in some of the most crucial physiological functions and are implicated in various pathologies. Another technique that is powerful to assess conformational dynamics is high-speed atomic force microscopy (HS-AFM), this approach has two significant advantages: (i) it is also a structural technique, meaning that it provides real-space real-time movies of molecules, and (ii) it operates under physiological and changeable conditions. Thus, the first advantage allows to characterize the structure and conformational changes of the channels at ~1nm lateral, ~0.1nm vertical and ~100ms temporal resolution. While the second advantage opens the experimental tool to the application of external stimuli, (bio)chemical and also, importantly, physical stimuli. In this project, we will develop novel extensions to HS- AFM to take movies of the conformational response of sensory channels to such physical cues. We will expose mechano-sensitive Piezo channels to force, temperature-sensitive TRPV channels to temperature- sweeps, and voltage-gated K+ channels to the direct application of transmembrane voltage, and image the structural changes of these proteins in response to such stimuli. This project will, on the one hand push the limits of HS-AFM technologically and create novel operational modalities of it and such further establish this rather new technology for a wide range of structure-function application in biomedical research, and on the other hand be transformative for the structural biology of sensory ion channels by providing insights into long-standing questions how these biological machines transform such physical stimuli into coordinated conformational dynamics that ultimately lead to channel gating.

Simon Scheuring，Weill Cornell Medicine 科学领域：6 MCB：分子和细胞生物学 / 5 IE：仪器和工程 项目摘要/摘要（文本30行）： 近年来，由于3D-的突破，我们在结构生物学方面取得了巨大进步 X射线衍射和改进的粒子分类算法的结晶方法和 开发冷冻EM的直接电子检测。结果，膜蛋白结构分辨率是 现在，常规并以每周近2个结构的速度进行。补充结构， 诸如FRET，EPR和HDX之类的技术提供了对动态和动力学范围的宝贵见解 构象状态。但是，所有实验性结构和动态技术都有一个盲点： 它们的适应性很差，可以根据物理刺激（例如力，温度和温度）分析蛋白质 电压。对于处理这些物理的感觉离子频道的情况，这尤其令人遗憾 刺激，因为它们参与了一些最关键的生理功能，并与 各种病理。另一种能够评估构象动力学强大的技术是高速 原子力显微镜（HS-AFM），该方法具有两个重要的优势：（i）它也是结构性的 技术，这意味着它提供了分子的真实空间实时电影，并且（ii）在 生理和可变条件。因此，第一个优势允许表征结构和 左侧约1nm处的通道的构象变化，垂直分辨率约为0.1nm和〜100ms。 虽然第二个优势为应用外部刺激的实验工具打开了 （生物）化学，重要的是物理刺激。在这个项目中，我们将为HS-开发新颖的扩展 AFM将感觉频道对这种物理提示的构象反应进行构象反应。我们将 暴露机械敏感的压电通道以强力，温度敏感的TRPV通道至温度 扫描和电压门控的K+通道直接应用跨膜电压，并为 这些蛋白质的结构变化响应于这种刺激。该项目将一方面推动 HS-AFM在技术上的限制并创造了它的新型操作方式，并进一步确定了这一点 在生物医学研究中以及在 另一只手通过提供洞察力来实现感官离子通道的结构生物学 这些生物机器如何将这种物理刺激转化为协调的问题 构象动力学最终导致通道门控。