Design of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins. Issue 1 (12th October 2015)

• Record Type: Journal Article
• Authors: Kamikawa, Yuko; Hori, Yuichiro; Yamashita, Kazuo; Jin, Lin; Hirayama, Shinya; Standley, Daron M.; Kikuchi, Kazuya
• Is Part Of: Chemical science. Volume 7:Issue 1(2016:Jan.)
• Page Start: 308
• Page End: 314
• Publication Date: 2015-10-12
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c5sc02351c
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : Quick and no-wash labeling of intracellular proteins was achieved in live cells using a PYP-tag mutant and a membrane-permeable fluorogenic probe with modular structures. Abstract : Conditional fluorescence imaging is a powerful technique for precise spatiotemporal analysis of proteins in live cells upon administration of a synthetic probe. To be applicable to various biological phenomena, probes must be membrane-permeable, have a high signal-to-noise ratio, and work quickly. To date, few probes meet all of these requirements. Here, we designed a fluorogenic probe (AcFCANB) that could label intracellular proteins fused to the photoactive yellow protein (PYP) tag in live cells within 30 min and used it to image heterochromatin protein 1 localization in nuclei. AcFCANB is based on a modular platform consisting of fluorophore, ligand and quencher. We accelerated the labeling reaction by strategic mutations of charged residues on the surface of PYP. A simple model based on molecular dynamics simulations quantitatively reproduced the cooperative effect of multiple mutations on labeling rate.