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

Simon Scheuring，Weill Cornell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，我们看到了结构生物学的巨大进步， X射线衍射的结晶方法和改进的颗粒分类算法， 低温电镜直接电子检测的发展。因此，膜蛋白结构分辨率是 现在相当常规，并以每周近2个结构的速度进展。为了补充结构， 技术，如FRET，EPR和HDX提供了宝贵的洞察力的动态和动力学的范围， 构象状态然而，所有的实验结构和动力学技术都有一个盲点： 它们不太适合于分析响应于物理刺激如力、温度和温度的蛋白质。 电压.这对于处理这些物理信号的感觉离子通道来说尤其令人遗憾。 刺激，因为它们参与了一些最重要的生理功能，并与 各种病理。另一种有效评估构象动力学的技术是高速电泳。 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实时空间实时电影，以及（ii）它在 生理和多变的条件。因此，第一个优点允许表征结构， 在~ 1 nm横向、~0.1nm纵向和~ 100 ms时间分辨率下观察通道的构象变化。 虽然第二个优点使实验工具对外部刺激的应用开放， （生物）化学的，重要的是，物理刺激。在这个项目中，我们将开发新的扩展HS- 原子力显微镜拍摄感觉通道对这些物理线索的构象反应的电影。我们将 将机械敏感的压电通道暴露于力，将温度敏感的TRPV通道暴露于温度- 扫描，电压门控K+通道直接施加跨膜电压，并成像 这些蛋白质的结构变化响应于这样的刺激。该项目将一方面推动 HS-AFM技术上的局限性，并创造了新的操作模式，从而进一步确立了这一点。 在生物医学研究中广泛的结构-功能应用的相当新的技术， 另一方面，通过提供以下见解， 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.