NEW CHEMICAL PROBES ENABLE MASS SPECTROMETRY-BASED FOOTPRINTING OF HUMAN PROTEIN STRUCTURE IN LIPID

新的化学探针实现了基于质谱的脂质中人类蛋白质结构的足迹

• 批准号: 10390166
• 负责人: MICHAEL L GROSS
• 金额: $ 25万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10390166
• 关键词: Cells; Cellular Structures; Chemicals; Chemistry; Coupled; Cryoelectron Microscopy; Crystallography; Data; Detection; Drug Targeting; Goals; Homeostasis; Human; Hydrophobicity; In Vitro; Iron; Label; Laboratories; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Mass Spectrum Analysis; Membrane; Membrane Lipids; Membrane Proteins; Membrane Transport Proteins; Methods; Modeling; Modernization; Motion; Physiological Processes; Proteins; Proteomics; Reagent; Reporting; Research Personnel; Resolution; Speed; Structure; System; base; improved; innovation; mass spectrometer; metal transporting protein 1; millisecond; physical property; protein structure; structural biology

The sensitivity, resolving power, and speed of modern mass spectrometers now afford the opportunity to develop bottom-up footprinting methods capable of resolving significant structural and dynamics questions of membrane proteins. This bottom-up approach is a fundamentally more powerful alternative to the top-down mass spectrometry (MS) studies that have been mainly limited to bacterial membrane proteins. We focus on human proteins because they participate in almost all physiological processes and represent more than 60% of drug targets. They, however, represent the most challenging targets for traditional high-resolution structural methods. Structures of about 100 of these proteins are known to date, leaving a large gap for footprinting MS to fill. Our long-term goal is to develop comprehensive footprinting MS methods that offer a unique approach to structure and dynamics of membrane proteins in live cells and in vitro lipid bilayers. Our objective here is to synthesize new chemical probes that provide high footprinting coverage to reveal the ligand interaction and dynamic transport motion of ferroportin, a model protein representing the largest superfamily of membrane transporters and maintaining iron homeostasis in humans. Our hypotheses are: (1) Complementary chemistry can maximize the coverage of footprinting and thereby improve its spatial resolution. Furthermore, tuning the physical properties of the labeling reagents will allow access to the hydrophobic region of membrane proteins. (2) Photoactivated fast footprinting can reveal dynamic transporter motions taking place within milliseconds, which is beyond the current scope of membrane structure biology. (3) Bio-orthogonal irreversible labeling can be optimized to reveal the cellular structure state of membrane proteins, a structure that is elusive by crystallography or cryo-EM. Use of these conventional methods requires purified proteins, but most membrane proteins are insufficiently stable to withstand demanding purification. Live-cell footprinting completely avoids this giant difficulty. Our hypotheses are built on extensive preliminary data produced in our laboratories. Specifically, we continue to demonstrate our capability to explore new chemistry and synthesize new reagents. Our ongoing studies prove the principle that MS footprinting can reveal ligand- binding interaction of human membrane proteins in lipid bilayer, and can report on their native structural state and motion in live cells. To accomplish our goals, we will pursue three specific aims: (1) develop new chemical probes to provide high footprinting coverage of membrane proteins; (2) implement the new probes in lipid membrane systems to study the ligand interaction and millisecond motion of ferroportin; and (3) demonstrate the new probes’ compatibility with live- cell footprinting by the detection of cellular motions and ligand interactions of ferroportin. Our innovative footprinting coupled with bottom-up MS proteomics analysis will establish effective, broad-based footprinting in live cells and lipid membranes. The significance of the proposed approach will expand because MS-based footprinting can be broadly applied by structural proteomics researchers to biomedically important human membrane proteins.

现代质谱仪的灵敏度、分辨率和速度现在可以满足 有机会开发自下而上的足迹方法，能够解决重大结构问题 和膜蛋白的动力学问题。这种自下而上的方法从根本上来说是一种更 自上而下的质谱（MS）研究的有力替代方案主要是 仅限于细菌膜蛋白。我们关注人类蛋白质，因为它们参与 几乎所有的生理过程，代表了 60% 以上的药物靶点。然而，他们 代表了传统高分辨率结构方法最具挑战性的目标。 迄今为止，其中约 100 种蛋白质的结构已为人所知，为足迹追踪留下了很大的空白 MS来补。我们的长期目标是开发全面的足迹 MS 方法，提供 研究活细胞和体外脂质膜蛋白结构和动力学的独特方法 双层。我们的目标是合成提供高足迹的新化学探针 覆盖揭示铁转运蛋白的配体相互作用和动态运输运动，这是一个模型 代表最大的膜转运蛋白超家族并维持铁的蛋白质 人类的体内平衡。我们的假设是：（1）互补化学可以最大化 覆盖足迹，从而提高其空间分辨率。此外，调整 标记试剂的物理特性将允许进入标记试剂的疏水区域 膜蛋白。 (2) 光激活快速足迹可以揭示动态转运蛋白运动 在几毫秒内发生，这超出了当前膜结构生物学的范围。 (3) 可以优化生物正交不可逆标记来揭示细胞结构状态 膜蛋白，一种通过晶体学或冷冻电镜难以捉摸的结构。使用这些 传统方法需要纯化蛋白质，但大多数膜蛋白不足以 稳定，可承受苛刻的净化。活细胞足迹完全避开了这个巨人 困难。我们的假设建立在我们实验室产生的大量初步数据的基础上。 具体来说，我们继续展示我们探索新化学和合成的能力 新试剂。我们正在进行的研究证明了 MS 足迹分析可以揭示配体的原理 脂双层中人膜蛋白的结合相互作用，并可以报告其天然的 活细胞的结构状态和运动。为了实现我们的目标，我们将追求三个具体目标 目标：（1）开发新的化学探针以提供膜的高足迹覆盖率 蛋白质； (2) 在脂膜系统中应用新探针来研究配体相互作用 和铁转运蛋白的毫秒运动； (3) 展示新探头与现场的兼容性 通过检测细胞运动和铁转运蛋白的配体相互作用来进行细胞足迹。我们的 创新足迹法与自下而上的 MS 蛋白质组学分析相结合将建立有效的、 活细胞和脂质膜中的广泛足迹。拟议的意义 方法将会扩展，因为基于 MS 的足迹可以广泛应用于结构 蛋白质组学研究人员研究了生物医学上重要的人类膜蛋白。