Quantitative label-free imaging of membrane protein interaction kinetics on cells

描述（由申请人提供）：嵌入或附着在细胞膜上的蛋白质执行许多至关重要的生物学功能，包括细胞信号，通信和重要物质进出细胞的运输。它们也是最重要的药物靶点和疾病生物标志物。尽管膜蛋白很重要，但研究膜蛋白，特别是量化它们与其他分子（如候选药物）的相互作用，一直是一项艰巨的挑战。目前的方法依赖于用荧光标签标记蛋白质或从其天然膜环境中提取蛋白质，然后纯化并固定在表面上进行结合动力学研究。前一种方法是终点分析，不能提供定量蛋白质相互作用所需的动力学信息，而后者不仅是劳动密集型的，而且容易发生天然结构的交替

DESCRIPTION (provided by applicant): Proteins embedded in or attached to cell membranes perform many critically important biological functions, including cell signaling, communication and the transport of vital substances into and out of cells. They are also the most important drug targets and disease biomarkers. Despite the importance, studying membrane proteins, especially quantifying their interactions with other molecules, such as drug candidates, has been a difficult challenge. Current methods rely on either labeling the proteins with fluorescent tags or extracting them from their native membrane environment, and then purifying and immobilizing them on a surface for binding kinetic studies. The former approach is an end-point-assay, which does not provide kinetics information required for quantifying protein interactions, while the latter is not only labor-intensive but also prone to alternation of the native structures and functions of the membrane proteins. This project focuses on developing a novel technique for studying and quantifying membrane protein interactions in their native cellular environment without the need of extraction, purification, or immobilization. The core of the technique is plasmonic-based electrical impedance microscopy (P-EIM) recently invented in the PIs' lab, which has several unique capabilities: 1) It is label free and can provide quantitative analysis of binding kinetics; 2) It has a high spatial resolution (sub-microns), and thus is suitable for analyzing membrane protein binding activities of single cells, and for mapping local binding kinetics of membrane proteins within a single cell; 3) It is fast (millisecond time resolution), which enables real-time tracking of cell signal transduction cascade triggered by small molecule binding to membrane proteins. Additionally, the technique allows for simultaneous plasmonic, impedance and fluorescence imaging, and combines the strengths of these methods in one system.