Revealing transmembrane conformational signaling through single-molecule FRET

通过单分子 FRET 揭示跨膜构象信号传导

• 批准号: 10552414
• 负责人: Gabriela Schlau-Cohen
• 金额: $ 42.82万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-21 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552414
• 关键词: Bacteria; Behavior; Biochemical; Biophysical Process; Biophysics; Cancer Biology; Cell membrane; Cell physiology; Chemoreceptors; Complex; Drug Design; Drug Targeting; Environment; Epidermal Growth Factor; Fluorescence; Individual; Investigation; Knowledge; Laboratories; Length; Ligands; Lipid Bilayers; Mammals; Membrane; Membrane Proteins; Methods; Molecular; Molecular Conformation; National Institute of General Medical Sciences; Organism; Physiological; Plants; Process; Proteins; Protocols documentation; Resolution; Role; Signal Transduction; Spectrum Analysis; Stimulus; Tars; Work; antibiotic resistant infections; cancer therapy; improved; insight; interest; microbial; nanodisk; non-Native; protein structure; receptor; single molecule; single-molecule FRET; sugar; temporal measurement; tool

Project Summary Membrane proteins regulate the cellular processes by which all organisms survive. Due to this crucial role, membrane proteins are 60% of drug targets. However, improvements to drug design are often impeded by open questions about their mechanisms. A fundamental function of membrane proteins is to transduce information across the membrane by encoding the presence of stimuli in their conformation. Therefore, knowledge of their conformations is required for the missing mechanistic understanding. However, high- resolution structural methods are often limited to individual domains and/or non-native conditions. In contrast, fluorescence-based single-molecule methods are amenable to physiological environments, yet can lack the spatial or temporal resolution required for key conformational changes. Our laboratory recently introduced new methods to improve the temporal resolution of single-molecule spectroscopy and, in the proposed work, will improve the spatial resolution. Investigations into transmembrane behaviors require the full-length protein structure, and thus its native membrane environment. Therefore, we have also developed robust protocols to solubilize membrane proteins from bacteria, plants, and mammals within discoidal lipid bilayers, known as nanodiscs. In initial studies, we used single-molecule spectroscopy and nanodiscs to reveal ligand-induced transmembrane conformational changes for two important receptors, the mammalian epidermal growth factor and the bacterial sugar chemoreceptor Tar. We are now primed to follow the propagation of ligand-induced conformational changes through the receptors and how these changes are controlled by the complex composition and organization of the plasma membrane. Altogether, this NIGMS MIRA application seeks to merge two of my laboratory's primary interests: (1) Developing and applying advanced single-molecule methods for molecular-level insight into protein machinery; and (2) Isolating and interrogating full-length membrane proteins in a near native environment using nanodiscs. Through this combination, we open a window into transmembrane conformational changes and the role of these conformations in cellular processes. Our contributions will impact fields ranging from single-molecule biophysics to cancer biology to microbial signaling.

项目摘要 膜蛋白调节所有有机体赖以生存的细胞过程。由于这一点至关重要 作用，膜蛋白是60%的药物靶点。然而，药物设计的改进常常受到阻碍。 通过对它们的机制提出公开的问题。膜蛋白的一个基本功能是转导 通过编码刺激在其构象中的存在来传递跨膜信息。因此， 对于缺失的机械性理解，需要了解它们的构象。然而，高- 解析结构方法通常限于单个域和/或非本地条件。相比之下， 基于荧光的单分子方法可以适应生理环境，但可能缺乏 关键构象变化所需的空间或时间分辨率。我们实验室最近引进了新的 提高单分子光谱学时间分辨率的方法，并在拟议的工作中，将 提高空间分辨率。跨膜行为的研究需要全长蛋白质 结构，因此它的天然膜环境。因此，我们还开发了强大的协议来 将细菌、植物和哺乳动物的膜蛋白溶解在盘状脂质双层中，称为 纳米盘。在最初的研究中，我们使用单分子光谱学和纳米盘来揭示配体诱导的 哺乳动物表皮生长因子两种重要受体的跨膜构象变化 细菌糖化学感受器焦油。我们现在准备好跟随配基诱导的 通过受体的构象变化以及这些变化是如何被复合体控制的 质膜的组成和组织。 总之，这个NIGMS Mira应用程序试图融合我的实验室的两个主要兴趣：(1) 开发和应用先进的单分子方法，在分子水平上深入了解蛋白质机制； 以及(2)使用纳米盘在接近自然的环境中分离和询问全长膜蛋白。 通过这种结合，我们打开了一扇窗，了解跨膜构象的变化和作用 这些构象在细胞过程中。我们的贡献将影响从单分子到 从生物物理学到癌症生物学再到微生物信号。