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）足迹正在成为回答生物学问题的强大手段 关于膜蛋白（MPS），几乎参与所有物理过程并代表更多 比60％的药物靶标。这种方法为动态的本地提供了足够的结构信息 MP在细胞中的构象和相互作用，这些细胞超出了传统结构方法的范围（例如， 冷冻EM和晶体学）。这种自下而上的MS足迹已完成，但可能会更多 与自上而下的本地MS相比，该MS不提供MPS的空间分辨率，并且在 非本地气相。在这里，我们建议继续开发活细胞中新型MS足迹方法 和本地膜。我们的目标是设计，准备，测试和改善提供高的化学问题 足迹覆盖范围。然后，我们将它们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，GLUT1，一种突出的抗癌药物靶标和约25％已知的MP 转运蛋白。然而，MPS的MS足迹提出了三个主要挑战。 1）国会议员 疏水并埋在脂质双层中，对传统探针有抵抗力（例如，HDX，•OH自由基） 穿透较差，标签不足。 2）跨膜区域的脂肪族侧链包含C – H 与大多数化学问题无反应的C-C键。 3）需要在细胞中进行足迹 或维持MP的天然构象和相互作用的机制。我们的假设是：（1）补充 自由基通过光化学和核螺旋试剂对C-H和X-H键的修改 最大化足迹覆盖范围。 （2）调整试剂或其前体的疏水性允许进入 到膜上的MPS。 （3）新型膜融合技术将惰性足迹引入活细胞 以及随后的光活化足迹的本地成员。我们的假设建立在广泛的基础上 初步数据。三年的资金支持出版在备受瞩目的期刊上的18篇论文。重要的 示例描述了连接到脂质体的TiO2纳米颗粒的激光激活以产生较高的局部 自由基的浓度。同时膜种群可以充分地进入足迹 结构分辨率报告了GLUT1的配体结合位点和摇杆开关运动。建立在这些基础上 成功，我们将追求两个具体的目标：（1）为MP的MS足迹开发新的化学问题；和 （2）在天然膜和活细胞中进行全面的足迹以揭示抗癌药物相互作用 和GLUT1的贩运法规。我们创新的足迹以及自下而上的MS蛋白质组学分析 将建立生物正交的足迹，以全面覆盖疏水性和亲水性 国会议员区域，揭示了在不可抑制天然的人类国会议员的药物相互作用和结构调节 设置。拟议方法的影响应很容易扩展，因为基于MS的足迹可能是 广泛应用于结构蛋白质组学，以加快细胞过程的药物发现和结构研究。