title: Redesign of a Fluorogenic Labeling System To Improve Surface Charge, Brightness, and Binding Kinetics for Imaging the Functional Localization of Bromodomains.
abstract: 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.
authors.affiliation: Graduate School of Engineering, Osaka University, Osaka 565-0871 (Japan).|IFReC, Osaka University, Osaka 565-0871 (Japan).|JST, PRESTO, Osaka 565-0871 (Japan).
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Hori
authors.affiliation: Graduate School of Engineering, Osaka University, Osaka 565-0871 (Japan).
authors.forename: Shinya
authors.initials: S
authors.lastname: Hirayama
authors.affiliation: Graduate School of Engineering, Osaka University, Osaka 565-0871 (Japan).
authors.forename: Motoki
authors.initials: M
authors.lastname: Sato
authors.affiliation: Graduate School of Engineering, Osaka University, Osaka 565-0871 (Japan). kkikuchi@mls.eng.osaka-u.ac.jp.|IFReC, Osaka University, Osaka 565-0871 (Japan). kkikuchi@mls.eng.osaka-u.ac.jp.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D001966:Bromine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006657:Histones / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D013499:Surface Properties
chemical_list: D005456:Fluorescent Dyes; D006657:Histones; D001966:Bromine
keywords: bromodomains; fluorescent probes; fluorogenic systems; protein labeling; protein modifications