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

西蒙·舍林，威尔·康奈尔医学公司 科学领域：6 MCB：分子和细胞生物学/5 IE：仪器与工程 项目摘要/摘要(30行文本)： 近年来，由于3D技术的突破，我们看到了结构生物学的巨大进步。 用于X射线衍射的结晶方法学和改进的颗粒分类算法 低温电子显微镜直接电子检测技术的发展。因此，膜蛋白结构的分辨率是 现在相当例行公事，以每周近2个结构的速度进步。为了补充结构， 像FRET、EPR和HDX这样的技术提供了对动力学和动力学范围的宝贵见解 构象状态。然而，所有实验性的结构和动力学技术都有一个盲点： 它们不太适合分析蛋白质对物理刺激的反应，如力、温度和 电压。这对于处理这些物理过程的感觉离子通道的情况来说尤其令人遗憾 刺激，因为它们参与了一些最关键的生理功能，并牵涉到 各种各样的病态。另一种评估构象动力学的强大技术是高速 原子力显微镜(HS-AFM)，这种方法有两个显著的优点：(I)它也是一种结构 技术，这意味着它提供了分子的真实空间实时电影，以及(Ii)它在 生理的和多变的条件。因此，第一个优点允许表征结构和 在横向分辨率为~1 nm、垂直分辨率为~0.1 nm、时间分辨率为~100ms的情况下，通道的构象变化。 虽然第二个优点为外部刺激的应用打开了实验工具， (生物)化学物质，还有重要的物理刺激。在这个项目中，我们将开发新的房协扩展部分- AFM拍摄感官通道对这种物理线索的构象反应的电影。我们会 将机械敏感的压电通道暴露在强迫下，温度敏感的TRPV通道暴露在温度- 扫描，和电压门控的K+通道直接施加跨膜电压，并成像 这些蛋白质对这种刺激的结构变化。这一项目一方面将推动 HS-AFM在技术上的局限性，并创造其新的操作模式等进一步确立这一点 相当新的技术，用于生物医学研究中广泛的结构功能应用，以及 另一方面，通过提供对以下方面的见解，将对感觉离子通道的结构生物学产生变革 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。

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.