Understanding membrane proteins’ allosteric modulation with cryo-EM

使用冷冻电镜了解膜蛋白变构调节

• 批准号: 10427240
• 负责人: Amedee des Georges
• 金额: $ 39.25万
• 依托单位: ADVANCED SCIENCE RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427240
• 关键词: Affect; Binding; Biological Models; Calcium; Classification; Cryoelectron Microscopy; Development; Disease; Drug Design; Family; G-Protein-Coupled Receptors; Heart; Image; Ion Channel; Ion Channel Gating; Ions; Knowledge; Laboratory Research; Lead; Ligands; Light; Lipids; Measurement; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; Muscle function; Mutagenesis; Output; Pathway interactions; Pharmaceutical Preparations; Play; Post-Translational Protein Processing; Process; Proteins; Regulation; Renal function; Role; Ryanodine Receptor Calcium Release Channel; Signal Transduction; System; Techniques; Testing; Transducers; Work; biophysical techniques; blood pressure regulation; design; drug development; heart function; interest; machine learning method; molecular dynamics; protein function; side effect; skeletal; small molecule; therapeutic protein; tool

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.

项目总结/摘要 我的实验室的研究重点是了解调控的分子机制， 膜蛋白功能和调节其输出信号。我们使用冷冻电镜和先进的 分类方法，如流形嵌入，结合建模和分子动力学， 研究配体如何产生构象平衡和偏置信号输出的变化。我们目前正在研究 这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， 膜张力传感的模型系统，可以产生有关基本的重要信息， 控制离子通道门控的原理以及脂质-蛋白质相互作用在此过程中的作用。 我们对兰尼碱受体（RyR）的机制和调节有着长期的兴趣，RyR是一种钙离子受体， 心脏和骨骼功能的基本释放通道。我们使用流形嵌入， 基于学习的方法来分析冷冻EM图像，以更好地了解小分子和离子如何 因为钙和ATP影响通道的构象能量景观。我们的目标是阐明 这个非常大的离子通道的门控机制，是理解蛋白质对其调节的先决条件 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低温电子显微镜，先进的 图像分类技术、建模和分子动力学模拟来描绘变构 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的门控和变构调节的分子理解， 离子通道在这方面取得的进展有可能为设计小型 分子变构调节剂，对它们的目标具有非常好的控制作用，有助于开发 副作用有限的药物。 长期目标是进一步开发和使用我们的工具，以更好地了解变构 调节其他类型的膜蛋白的治疗重要性。特别是G蛋白偶联 受体配体可以在称为偏置信号传导的过程中诱导不同转换器的选择性结合。 在这一过程中发挥作用的分子机制仍然知之甚少，尽管它们具有基本的 开发更安全的药物。