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: 10442739
• 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/10442739
• 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.

西蒙·舍林，威尔康奈尔医学 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（30 行文本）： 近年来，由于 3D 技术的突破，我们看到结构生物学取得了巨大进展。 X 射线衍射结晶方法和改进的颗粒分类算法以及 冷冻电镜直接电子检测的发展。因此，膜蛋白结构分辨率为 现在相当常规，并且以每周几乎 2 个结构的速度进行。为了补充结构， FRET、EPR 和 HDX 等技术为了解物体的动力学和动力学范围提供了宝贵的见解 构象状态。然而，所有实验结构和动力学技术都有一个盲点： 它们不适合分析蛋白质对物理刺激（如力、温度和 电压。对于处理这些物理的感觉离子通道来说，这是特别令人遗憾的。 刺激，因为它们涉及一些最重要的生理功能，并与 各种病理。另一种强大的评估构象动力学的技术是高速 原子力显微镜（HS-AFM），这种方法有两个显着的优点：（i）它也是一种结构 技术，这意味着它提供分子的真实空间实时电影，并且（ii）它在 生理和多变的条件。因此，第一个优点允许表征结构和 通道的构象变化在〜1nm横向，〜0.1nm垂直和〜100ms时间分辨率。 虽然第二个优点使实验工具能够应用外部刺激， （生物）化学刺激，而且重要的是物理刺激。在这个项目中，我们将开发 HS- 的新颖扩展 AFM 可以拍摄感觉通道对此类物理线索的构象反应的电影。我们将 暴露机械敏感的压电通道，以迫使温度敏感的 TRPV 通道受到温度影响 扫描和电压门控 K+ 通道直接应用跨膜电压，并对 这些蛋白质响应此类刺激而发生结构变化。该项目一方面将推动 HS-AFM 在技术上的局限性并创造了它的新颖的操作模式，从而进一步确立了这一点 在生物医学研究中广泛的结构功能应用的新技术 另一方面，通过提供对感觉离子通道结构生物学的变革 长期存在的问题是这些生物机器如何将这种物理刺激转化为协调的 最终导致通道门控的构象动力学。