Ayele Tewoderos M, Knutson Steve D, Heemstra Jennifer M
Department of Chemistry, Emory University, Atlanta, GA, United States.
Department of Chemistry, Emory University, Atlanta, GA, United States.
Methods Enzymol. 2020;639:355-377. doi: 10.1016/bs.mie.2020.04.019. Epub 2020 Apr 28.
Fluorescence microscopy has dramatically advanced our understanding of the processes that drive biological systems by enabling the imaging and tracking of biomolecules of interest inside of living cells. In particular, proteins of interest can be genetically tagged with fluorescent proteins or labeled with small molecule fluorophore probes to enable visualization. However, both of these methods are generally limited in signal-to-background resolution and options are limited for achieving temporal control over labeling. Photoreactive "fluorogenic" dyes can overcome these limitations and enable user-defined crosslinking with low background fluorescence. In this chapter, we discuss current approaches for live cell protein labeling with particular emphasis on the novel use of photoreactive fluorogenic dyes for protein imaging. We further describe in detail the synthesis and characterization of a fluorogenic malachite green probe functionalized with a photoreactive diazirine crosslinker and illustrate how to apply this probe toward covalent photoaffinity labeling and imaging of target proteins in live cells.
荧光显微镜通过对活细胞内感兴趣的生物分子进行成像和追踪，极大地推进了我们对驱动生物系统过程的理解。特别是，感兴趣的蛋白质可以用荧光蛋白进行基因标记，或用小分子荧光团探针进行标记以实现可视化。然而，这两种方法在信号与背景分辨率方面通常都有局限，并且在实现对标记的时间控制方面选择有限。光反应性“荧光生成”染料可以克服这些限制，并能以低背景荧光实现用户定义的交联。在本章中，我们将讨论活细胞蛋白质标记的当前方法，特别强调光反应性荧光生成染料在蛋白质成像中的新应用。我们还将详细描述一种用光反应性重氮交联剂功能化的荧光孔雀石绿探针的合成与表征，并说明如何将该探针应用于活细胞中靶蛋白的共价光亲和标记和成像。
