title: Chromophore-assisted light inactivation of HaloTag fusion proteins labeled with eosin in living cells.
abstract: Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen ((1)O(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of ((1)O(2)) than fluorescein and about 5-fold efficiency in CALI of β-galactosidase by using an eosin-labeled anti-β-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.
authors.forename: Kiwamu
authors.initials: K
authors.lastname: Takemoto
authors.forename: Tomoki
authors.initials: T
authors.lastname: Matsuda
authors.forename: Mark
authors.initials: M
authors.lastname: McDougall
authors.forename: Dieter H
authors.initials: DH
authors.lastname: Klaubert
authors.forename: Akira
authors.initials: A
authors.lastname: Hasegawa
authors.forename: Georgyi V
authors.initials: GV
authors.lastname: Los
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
mesh_terms: D064107:Aurora Kinase B; D064127:Aurora Kinases; D004801:Eosine Yellowish-(YS) / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D008024:Ligands; D008027:Light; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011493:Protein Kinase C / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000528:radiation effects; D026082:Singlet Oxygen; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000528:radiation effects
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D017319:Photosensitizing Agents; D026082:Singlet Oxygen; C087857:protein kinase C gamma; C576921:AURKB protein, human; D064107:Aurora Kinase B; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D001616:beta-Galactosidase; D004801:Eosine Yellowish-(YS)