title: Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.
abstract: Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.
authors: School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School , Shenzhen 518055, China.|Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University , New York, New York 10065, United States.
authors: Tao
authors: T
authors: Peng
authors: Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University , New York, New York 10065, United States.
authors: Howard C
authors: HC
authors: Hang
mesh_terms: D001665:Binding Sites; D002470:Cell Survival; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D061848:Optical Imaging / Q000379:methods*; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: C080082:IFITM3 protein, human; D008565:Membrane Proteins; D016601:RNA-Binding Proteins