title: Fluorophore labeling of native FKBP12 by ligand-directed tosyl chemistry allows detection of its molecular interactions in vitro and in living cells.
abstract: Introducing synthetic fluorophores into specific endogenous proteins and analyzing their function in living cells are a great challenge in chemical biology. Toward this end, we demonstrate the target-selective and site-specific fluorescent labeling of native FKBP12 (FK506-binding protein 12) in vitro and in living cells using ligand-directed tosyl (LDT) chemistry. The LDT-mediated labeling yielded a semisynthetic FKBP12 containing the Oregon green (OG) dye near the catalytic pocket. The OG-labeled FKBP12 (OG-FKBP12) acted as a fluorescent reporter that allows monitoring of its interaction with rapamycin and FRB (FKBP-rapamycin-binding domain) in vitro. We also successfully demonstrated the visualization of the rapamycin-mediated complexation of the OG-FKBP12 and FRB inside of living cells by the combined use with fluorescent protein-tag technology and Förster resonance energy-transfer imaging.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Tamura
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kioi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Miki
authors.forename: Shinya
authors.initials: S
authors.lastname: Tsukiji
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
mesh_terms: D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D015394:Molecular Structure; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D022061:Tacrolimus Binding Protein 1A