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Redesign of a Fluorogenic Labeling System To Improve Surface Charge, Brightness, and Binding Kinetics for Imaging the Functional Localization of Bromodomains
Author(s) -
Hori Yuichiro,
Hirayama Shinya,
Sato Motoki,
Kikuchi Kazuya
Publication year - 2015
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201506935
Subject(s) - bromodomain , fluorescence , biophysics , kinetics , chemistry , mutant , in vitro , fluorescence lifetime imaging microscopy , microbiology and biotechnology , biochemistry , biology , histone , physics , quantum mechanics , gene
Protein labeling with fluorogenic probes is a powerful method for the imaging of cellular proteins. The labeling time and fluorescence contrast of the fluorogenic probes are critical factors for the precise spatiotemporal imaging of protein dynamics in living cells. To address these issues, we took mutational and chemical approaches to increase the labeling kinetics and fluorescence intensity of fluorogenic PYP‐tag probes. Because of charge‐reversal mutations in PYP‐tag and probe redesign, the labeling reaction was accelerated by a factor of 18 in vitro, and intracellular proteins were detected with an incubation period of only 1 min. The brightness of the probe both in vitro and in living cells was enhanced by the mutant tag. Furthermore, we applied this system to the imaging analysis of bromodomains. The labeled mutant tag successfully detected the localization of bromodomains to acetylhistone and the disruption of the bromodomain–acetylhistone interaction by a bromodomain inhibitor.