Department of Chemistry, University of Ottawa, 10 Marie-Curie, Ottawa, ON K1N 6N5 (Canada).
Angew Chem Int Ed Engl. 2014 Dec 8;53(50):13785-8. doi: 10.1002/anie.201408015. Epub 2014 Oct 14.
A fluorescent protein-labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.
开发了一种荧光蛋白标记策略，其中感兴趣的蛋白质（POI）通过短肽序列进行基因标记，该短肽序列带有两个半胱氨酸残基，可选择性地与合成的荧光发生试剂反应。这些荧光发生试剂包括荧光团和两个马来酰亚胺基团，在标记反应过程中，它们都通过巯基加成反应来猝灭荧光。新合成的荧光发生试剂进行了动力学表征，以证明马来酰亚胺上甲氧基取代基的重要性，该取代基可抑制与谷胱甘肽（一种细胞内巯基）的反应性，同时保持与二硫键标签的反应性。该系统允许快速且特异性地标记细胞内的 POI。
