title: Ligand-directed acyl imidazole chemistry for labeling of membrane-bound proteins on live cells.
abstract: Chemistry-based protein labeling in living cells is undoubtedly useful for understanding natural protein functions and for biological/pharmaceutical applications. Here, we report a novel approach for endogenous membrane-bound protein labeling for both in vitro and live cell conditions. A moderately reactive alkyloxyacyl imidazole (AI) assisted by ligand-binding affinity (ligand-directed AI (LDAI)) chemistry allowed us to selectively modify natural proteins, such as dihydrofolate reductase (DHFR) and folate receptor (FR), neither of which could be efficiently labeled using the recently developed ligand-directed tosylate approach. It was clear that LDAI selectively labeled a single Lys(K32) in DHFR, proximal to the ligand-binding pocket. We also demonstrate that the fluorescein-labeled (endogenous, by LDAI) FR works as a fluorescent biosensor on the live KB cell surface, which allowed us to carry out unprecedented in situ kinetic analysis of ligand binding to FR.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors.forename: Sho-hei
authors.initials: SH
authors.lastname: Fujishima
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Yasui
authors.forename: Takayuki
authors.initials: T
authors.lastname: Miki
authors.forename: Akio
authors.initials: A
authors.lastname: Ojida
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
mesh_terms: D005453:Fluorescence; D058973:Folate Receptors, GPI-Anchored / Q000737:chemistry*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry*; D007624:KB Cells; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D015394:Molecular Structure; D013194:Staining and Labeling; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D058973:Folate Receptors, GPI-Anchored; D007093:Imidazoles; D008024:Ligands; D008565:Membrane Proteins; D013762:Tetrahydrofolate Dehydrogenase