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

项目摘要 基于质谱学(MS)的足迹技术正在成为回答生物问题的有力手段 关于膜蛋白(MPS)，它参与几乎所有的生理过程，代表更多 超过60%的药物靶标。这种方法为动态的、本机的 MPS在细胞中的构象和相互作用，这超出了传统结构方法的范围(例如， 低温电子显微镜和结晶学)。这种自下而上的MS足迹是对MS足迹的补充，但可能会更多 比自上而下的原生MS更具信息性，后者不为MPS提供空间分辨率，并在 非本地气相。在这里，我们建议继续开发活细胞中MS足迹的新方法 和天然的膜。我们的目标是设计、准备、测试和改进化学探针，以提供高质量的 足迹覆盖范围。然后我们将应用它们来揭示药物相互作用和细胞贩运的调节 葡萄糖转运蛋白，GLUT1，一个重要的抗癌药物靶点和模型MP，代表了已知的约25% 运输蛋白。然而，议员的MS足迹构成了三大挑战。1)MPS，即 疏水性和埋藏在脂质双层中，对传统的探针(例如，HDX，·OH自由基)具有抵抗力 穿透性差，标签不充分。2)跨膜区的脂肪族侧链含有C-H 以及与大多数化学探针不起反应的C-C键。3)需要在单元格中进行足迹 或膜以维持MPS的天然构象和相互作用。我们的假设是：(1)互补性 光化学和亲核试剂产生的自由基对C-H和X-H键的修饰 最大限度地扩大覆盖范围。(2)调节试剂或其前体的疏水性允许访问 到膜包埋的MPS。(3)新的膜融合技术将惰性脚印引入活细胞 以及用于后续光激活足迹的天然膜。我们的假设建立在广泛的基础上 初步数据。三年的资金支持在知名期刊上发表了18篇论文。一个重要的 实例描述了激光激活附着到脂质体上的二氧化钛纳米颗粒以产生高局部 自由基的浓度。同时膜穿孔允许自由基以足够的速度进入足迹 报告GLUT1的配体结合位置和摇杆开关运动的结构分辨率。建立在这些基础上 为了取得成功，我们将追求两个具体目标：(1)为MPS的MS足迹开发新的化学探针；以及 (2)在天然膜和活细胞中进行全面的足迹研究，以揭示抗癌药物的相互作用 和GLUT1的贩运规定。我们的创新足迹与自下而上的MS蛋白质组分析相结合 将建立生物正交足迹打印机，全面覆盖疏水和亲水 MPS的区域和揭示药物相互作用和无可争辩的天然条件下人MPS的结构调节 设置。建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，以加速药物发现和细胞过程的结构研究。

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.