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.

项目总结