Redesign of a Fluorogenic Labeling System To Improve Surface Charge, Brightness, and Binding Kinetics for Imaging the Functional Localization of Bromodomains.
Angew Chem Int Ed Engl
; 54(48): 14368-71, 2015 Nov 23.
Article
in En
| MEDLINE
| ID: mdl-26434386
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 1â
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Fluorescent Dyes
Language:
En
Journal:
Angew Chem Int Ed Engl
Year:
2015
Document type:
Article