Department of Chemistry and Biochemistry, University of Notre Dame , Notre Dame, Indiana 46556, United States.
Anal Chem. 2017 Sep 5;89(17):9091-9099. doi: 10.1021/acs.analchem.7b01796. Epub 2017 Aug 22.
The specific interaction between a ligand and a protein is a key component in minimizing off-target effects in drug discovery. Investigating these interactions with membrane protein receptors can be quite challenging. In this report, we show how spectral variance observed in surface-enhanced Raman scattering (SERS) and tip-enhanced Raman scattering (TERS) can be correlated with ligand specificity in affinity-based assays. Variations in the enhanced Raman spectra of three peptide ligands (i.e., cyclic-RGDFC, cyclic-isoDGRFC, and CisoDGRC), which have different binding affinity to αvβ3 integrin, are reported from isolated proteins and from receptors in intact cancer cell membranes. The SERS signal from the purified proteins provides basis spectra to analyze the signals in cells. Differences in the spectral variance within the SERS and TERS data for each ligand indicate larger variance for nonspecific ligand-receptor interactions. The SERS and TERS results are correlated with single particle tracking experiments of the ligand-functionalized nanoparticles with purified receptors on glass surfaces and living cells. These results demonstrate the ability to elucidate protein-ligand recognition using the observed vibrational spectra and provide perspective on binding specificity for small-molecule ligands in intact cell membranes, demonstrating a new approach for investigating drug specificity.
配体与蛋白质之间的特定相互作用是药物发现中最小化非靶标效应的关键组成部分。研究这些与膜蛋白受体的相互作用具有相当大的挑战性。在本报告中，我们展示了如何将表面增强拉曼散射（SERS）和尖端增强拉曼散射（TERS）中观察到的光谱方差与基于亲和力的测定中的配体特异性相关联。报告了三种肽配体（即环状-RGDFC、环状-isoDGRFC 和 CisoDGRC）的增强拉曼光谱的变化，它们与αvβ3 整合素的结合亲和力不同，从分离的蛋白质和完整细胞膜中的受体中得到。从纯化蛋白中得到的 SERS 信号为分析细胞中的信号提供了基础光谱。每个配体的 SERS 和 TERS 数据内的光谱方差差异表明非特异性配体-受体相互作用的方差更大。SERS 和 TERS 的结果与玻璃表面上纯化受体和活细胞中配体功能化纳米粒子的单粒子跟踪实验相关联。这些结果证明了使用观察到的振动光谱阐明蛋白质-配体识别的能力，并为完整细胞膜中小分子配体的结合特异性提供了视角，展示了一种研究药物特异性的新方法。
