Understanding membrane proteins’ allosteric modulation with cryo-EM

项目摘要/摘要 我的实验室的研究重点是了解调控的关键分子机制 膜蛋白的功能及其对其输出信号的调制。我们使用低温电磁和先进的 分类方法，如流形嵌入，结合建模和分子动力学 研究配体如何在构象平衡和偏向信号输出中产生位移。我们目前正在研究 两个系统使我们能够研究不同类型的配体如何影响离子通道的门控。 我们正在研究脂质在触发MSCs家族机械敏感通道门控中的作用，a 薄膜张力传感的模型系统，可提供有关基本原理的重要信息 管理离子通道门控的原理以及脂质-蛋白质相互作用在这一过程中所起的作用。 长期以来，我们一直对兰尼定受体(RyR)的机制和调节感兴趣，RyR是一种钙 释放心脏和骨骼功能的基本通道。我们使用的是流形嵌入，一种机器 基于学习的方法来分析低温电子显微镜图像，以更好地了解小分子和离子等 因为钙和三磷酸腺苷影响通道的构象能量格局。我们的目标是阐明 这一非常大的离子通道的门控机制，是理解蛋白质对其调控的先决条件 配体、翻译后修饰和药物。 我们解决这些基本问题的方法是使用冷冻电子显微镜的组合，先进的 用于描述变构的图像分类技术、建模和分子动力学模拟 路径，然后用生物物理方法(如单通道)测试所提出的模型 测量、HDX-MS和诱变。 我们的最终目标是极大地增加我们对门控和变构调节的分子理解 离子通道。在这些知识方面的进步有可能为小型汽车的设计开辟道路 分子变构调节剂对其靶标具有很好的可控性，有助于开发 副作用有限的药物。 一个更长期的目标是进一步开发和使用我们的工具来更好地了解变构 调节其他种类的膜蛋白具有重要的治疗作用。特别是，G蛋白偶联 受体配体可以诱导不同转导分子的选择性结合，这一过程称为有偏见的信号转导。 在这一过程中发挥作用的分子机制仍然知之甚少，尽管它们的基本原理 这对开发更安全的药物具有重要意义。

Project Summary/Abstract My laboratory's research is focused on understanding the molecular mechanisms key to the regulation of membrane protein function and to the modulation of their output signaling. We use cryo-EM and advanced classification methods, such as manifold embedding, combined with modeling and molecular dynamics to study how ligands produce shifts in conformation equilibria and bias signaling output. We are currently studying two systems that allow us to study how different types of ligands influence the gating of ion channels. We are studying the role of lipids in triggering gating of mechanosensitive channels of the MscS family, a model system of membrane tension-sensing which can yield important information about the fundamental principles governing ion channel gating and the role that lipid-protein interactions play in this process. We have a longstanding interest in the mechanism and modulation of the ryanodine receptor (RyR), a calcium release channel fundamental to heart and skeletal function. We are using manifold embedding, a machine learning-based method to analyze cryo-EM images, to better understand how small molecules and ions such as calcium and ATP affect the conformational energy landscape of the channel. We aim to shed light on the gating mechanism of this very large ion channel, a prerequisite to understanding of its modulation by protein ligands, post-translational modifications and drugs. Our approach to these fundamental problems is to use a commbination of cryo-electron microscopy, advanced image classification techniques, modelling and molecular dynamics simulations to delineate allosteric pathways mechanistically and then test proposed models with biophysical methods such as single-channel measurements, HDX-MS and mutagenesis. Our ultimate aim is to greatly increase our molecular understanding of the gating and allosteric modulation of ion channels. Progress towards such knowledge has the potential to open the way for the design of small molecule allosteric modulators with very well controlled effects on their targets, aiding the development of drugs with limited side effects. A longer-term aim is to further develp and use our tools towards gaining a better understanding of allosteric modulation in other classes of membrane proteins of therapeutic importance. In particular, G protein-coupled receptor ligands can induce the selective binding of different transducers in a process called biased signaling. The molecular mechanisms at play in this process are still poorly understood despite their fundamental importance for the development of safer drugs.