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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要基于质谱 (MS) 的足迹分析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这种方法为动态、天然的药物靶标提供了足够的结构信息。细胞内 MP 的构象和相互作用，这是传统结构方法无法实现的（例如，这种自下而上的 MS 足迹是互补的，但可能更多。比自上而下的本地 MS 信息更丰富，后者不为 MP 提供空间分辨率，并且是在在这里，我们建议继续开发活细胞中的新型 MS 足迹方法。我们的目标是设计、制备、测试和改进提供高通量的化学探针。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 GLUT1，一个重要的抗癌药物靶点和一个模型 MP，约占已知的 25% 然而，MP 的转运蛋白的 MS 足迹提出了三个主要挑战：1) MP。疏水性并埋在脂质双层中，对传统探针（例如 HDX、·OH 自由基）具有抵抗力 2) 跨膜区的脂肪族侧链含有C–H 和与大多数化学探针不发生反应的 C-C 键 3) 足迹需要在细胞中进行。我们的假设是：（1）互补。通过光化学产生的自由基和亲核试剂对 C-H 和 X-H 键进行修饰 (2) 调节试剂或其前体的疏水性可以允许访问 (3) 新型膜融合技术将惰性足迹引入活细胞中。以及用于后续光激活足迹的天然膜。我们的假设是建立在广泛的基础上的。初步数据。三年的资助支持在知名期刊上发表了 18 篇论文。示例描述了激光激活附着在脂质体上的 TiO2 纳米粒子，以产生高局部同时膜穿孔允许自由基以足够的量进入足迹。报告配体结合位点和 GLUT1 摇杆开关运动的结构分辨率。如果取得成功，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) 对天然膜和活细胞进行全面的足迹分析，以揭示抗癌药物的相互作用我们的创新足迹与自下而上的 MS 蛋白质组学分析相结合。将建立生物正交足迹，全面覆盖疏水性和亲水性 MP 区域并揭示药物相互作用和人类 MP 在无可争议的天然条件下的结构调节所提出的方法的影响应该很容易扩大，因为基于 MS 的足迹可以被扩展。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。