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

新的化学探针能够对脂质膜和细胞中的人体蛋白质结构进行基于质谱的足迹分析

• 批准号: 10587527
• 负责人: MICHAEL L GROSS
• 金额: $ 46.57万
• 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 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587527
• 关键词: Antineoplastic Agents; Azides; Benchmarking; Benzophenones; Binding Sites; Biological; Carrier Proteins; Cell Membrane Permeability; Cell Separation; Cell Survival; Cell physiology; Cells; Cellular Metabolic Process; Chemical Structure; Chemicals; Chemistry; Consumption; Coupled; Cryoelectron Microscopy; Crystallography; Data; Development; Drug Interactions; Drug Targeting; Ensure; FASTK Gene; Free Radicals; Funding; Gases; Glucose Transporter; Goals; Grant; Human; Human body; Hydrogen Bonding; Hydrophobicity; Iodides; Iodine; Journals; Label; Lasers; Ligand Binding; Lipid Bilayers; Lipids; Liposomes; Mass Spectrum Analysis; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Membrane Transport Proteins; Methodology; Methods; Modification; Molecular Conformation; Monitor; Motion; Paper; Penetration; Permeability; Peroxides; Phase; Physiological Processes; Process; Protein Dynamics; Protein Footprinting; Proteins; Proteomics; Public Health; Publications; R24; Reaction; Reactive Oxygen Species; Reagent; Regulation; Reporting; Research; Resistance; Resolution; Resources; SLC2A1 gene; SNAP receptor; Series; Side; Structure; Techniques; Technology; Testing; Time; Transmembrane Domain; United States National Institutes of Health; blood glucose regulation; carbene; design; drug candidate; drug discovery; glucose-regulated proteins; hydrophilicity; improved; innovation; lipid solubility; materials science; membrane model; nanodisk; nanoparticle; non-Native; novel; physical property; protein purification; protein structure; reconstitution; structural biology; success; titanium dioxide; tool; trafficking

Project Summary Mass spectrometry (MS) based footprinting is emerging as a powerful means to answer biological questions about membrane proteins (MPs), which participate in almost all physiological processes and represent more than 60% of drug targets. This approach affords sufficient structural information for the dynamic, native conformations and interactions of MPs in cells, which are beyond the reach of traditional structural methods (e.g., cryo-EM and crystallography). This bottom-up MS footprinting is complementary to but potentially more informative than top-down native MS, which does not provide spatial resolution for MPs and is conducted in the nonnative gas phase. Here we propose to continue development of novel MS footprinting methods in live cells and native membranes. Our objective is to design, prepare, test, and improve chemical probes that provide high footprinting coverage. We will then apply them to reveal drug interactions and cellular trafficking regulation of a glucose transporter, GLUT1, a prominent anticancer drug target and a model MP representing ~ 25% of known transport proteins. MS footprinting of MPs, however, poses three major challenges. 1) MPs, which are hydrophobic and buried in lipid bilayers, are resistant to traditional probes (e.g., HDX, •OH radicals) that penetrate poorly and give insufficient labeling. 2) Aliphatic side chains of transmembrane regions contain C–H and C-C bonds that are unreactive with most chemical probes. 3) The footprinting needs to be conducted in cells or membranes to maintain native conformation and interaction of MPs. Our hypotheses are: (1) Complementary modifications of C-H and X–H bonds by free radicals produced photochemically and by nucleophilic reagents maximize footprinting coverage. (2) Tuning the hydrophobicity of the reagents or their precursors allows access to membrane-embedded MPs. (3) Novel membrane fusion techniques introduce inert footprinters into live cells and native membranes for subsequent photoactivated footprinting. Our hypotheses are built on extensive preliminary data. Three years of funding supported publication of 18 papers in high-profile journals. A significant example describes laser activation of TiO2 nanoparticles attached to liposomes to generate high local concentrations of radicals. Simultaneous membrane poration permits radical entry to footprint with sufficient structural resolution that reports the ligand-binding sites and rocker-switch motions of GLUT1. Building on these successes, we will pursue two specific aims: (1) develop new chemical probes for MS footprinting of MPs; and (2) conduct comprehensive footprinting in native membranes and live cells to reveal anticancer drug interactions and trafficking regulations of GLUT1. Our innovative footprinting coupled with bottom-up MS proteomics analysis will establish bio-orthogonal footprinters that afford comprehensive coverage of both hydrophobic and hydrophilic regions of MPs and reveal drug interactions and structural regulation of human MPs under inarguable native settings. The impact of the proposed approach should readily expand because MS-based footprinting can be broadly applied in structural proteomics to expedite drug discovery and structural studies of cellular processes.

项目摘要 基于质谱（MS）的足迹正在成为回答生物学问题的有力手段 关于膜蛋白（MP），它参与几乎所有的生理过程，并代表更多 超过60%的药物靶点。这种方法提供了足够的结构信息的动态，本地 细胞中MP的构象和相互作用，这超出了传统结构方法的范围（例如， cryo-EM和晶体学）。这种自下而上的微软足迹是补充，但可能更多 信息比自上而下的本地MS，它不提供MP的空间分辨率，并在 非天然气相在这里，我们建议继续开发新的MS足迹法在活细胞中 和天然膜。我们的目标是设计、制备、测试和改进化学探针， 足迹覆盖。然后，我们将应用它们来揭示药物相互作用和细胞运输调节， 葡萄糖转运蛋白GLUT 1是一种重要的抗癌药物靶点，也是一种模型MP，占已知MP的约25%。 转运蛋白然而，议员们的MS足迹构成了三大挑战。1)MP，即 疏水性的并且埋在脂质双层中，对传统的探针有抗性（例如，HDX，·OH自由基）， 渗透性差，标记不充分。2)跨膜区的脂肪族侧链含有C-H 和不与大多数化学探针反应的C-C键。3)足迹需要在细胞中进行 或膜以保持MP的天然构象和相互作用。我们的假设是：（1）互补性 通过光化学产生的自由基和亲核试剂对C-H键和X-H键的修饰 最大化足迹覆盖范围。(2)调节试剂或其前体的疏水性允许进入 到膜包埋的MP。(3)新型膜融合技术将惰性足迹引入活细胞 和用于随后的光活化足迹的天然膜。我们的假设是建立在广泛的 初步数据。三年的资助支持了18篇论文在知名期刊上发表。显著 实施例描述了附着于脂质体的TiO 2纳米颗粒的激光活化，以产生高局部 自由基的浓度。同时膜穿孔允许自由基进入足迹， 结构解析，报告GLUT 1的配体结合位点和摇杆开关运动。根据这些 成功后，我们将追求两个具体目标：（1）开发新的化学探针用于MP的MS足迹; (2)在天然膜和活细胞中进行全面的足迹分析，以揭示抗癌药物的相互作用 和GLUT 1的贩运法规。我们创新的足迹法结合自下而上的MS蛋白质组学分析 将建立生物正交足迹，提供全面覆盖的疏水和亲水 区域的MPs和揭示药物相互作用和结构调控的人MPs下不可否认的天然 设置.所提议的方法的影响应该很容易扩大，因为基于MS的足迹可以 广泛应用于结构蛋白质组学，以加快药物发现和细胞过程的结构研究。