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 膜蛋白调节所有生物存活的细胞过程。由于这个关键 角色，膜蛋白是药物靶标的60％。但是，通常会阻碍药物设计的改进 通过公开有关其机制的问题。膜蛋白的基本功能是转导 通过编码刺激在其构象中的存在来跨膜的信息。所以， 缺失的机械理解需要了解其构象。但是，高 - 分辨率结构方法通常仅限于单个领域和/或非本地条件。相比之下， 基于荧光的单分子方法适合生理环境，但可能缺乏 关键构象变化所需的空间或时间分辨率。我们的实验室最近推出了新的 改善单分子光谱法以及在拟议的工作中的时间分辨率的方法将 改善空间分辨率。对跨膜行为的研究需要全长蛋白 结构，因此其本地膜环境。因此，我们还开发了强大的协议 从细菌，植物和哺乳动物溶解的膜蛋白中溶解盘状脂质双层中的膜蛋白，称为 纳米盘。在最初的研究中，我们使用单分子光谱和纳米散发揭示了配体诱导的 两个重要受体的跨膜构象变化，哺乳动物表皮生长因子 和细菌糖化学感受器焦油。现在，我们正在努力遵循配体诱导的传播 通过受体的构象变化以及如何由复合物控制这些变化 质膜的组成和组织。 总的来说，此Nigms Mira应用程序旨在合并我的两个实验室的主要利益：（1） 开发和应用高级单分子方法，以洞悉蛋白质机械的分子水平； （2）使用纳米盘在接近天然的环境中隔离和询问全长膜蛋白。 通过这种组合，我们为跨膜构象变化打开了一个窗口 这些在细胞过程中的构象。我们的贡献将影响从单分子 生物物理学对微生物信号传导的生物物理学。