Revealing transmembrane conformational signaling through single-molecule FRET

项目摘要 膜蛋白调节所有生物体赖以生存的细胞过程。由于这一关键 膜蛋白是60%的药物靶点。然而，药物设计的改进往往受到阻碍， 对它们的机制提出了公开的问题。膜蛋白的一个基本功能是 通过编码刺激物构象中的存在，跨膜传递信息。因此，我们认为， 需要了解它们的构象以获得缺失的机械理解。然而，高- 解析结构方法通常限于单个结构域和/或非天然条件。与此相反， 基于荧光的单分子方法适用于生理环境，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 方法，以提高单分子光谱的时间分辨率，并在拟议的工作，将 提高空间分辨率。跨膜行为的研究需要全长蛋白 结构，因此其天然膜环境。因此，我们还开发了强大的协议， 将来自细菌、植物和哺乳动物膜蛋白溶解在盘状脂质双层内，称为 纳米盘在最初的研究中，我们使用单分子光谱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 和细菌糖化学感受器焦油。我们现在准备跟踪配体诱导的 通过受体的构象变化，以及这些变化如何由复合物控制 质膜的组成和组织。 总而言之，这个NIGMS MIRA应用程序旨在合并我实验室的两个主要利益：（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; 和（2）使用纳米盘在接近天然的环境中分离和询问全长膜蛋白。 通过这种结合，我们打开了一扇了解跨膜构象变化和 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。

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.