Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

• Published in: Nature communications Vol. 11; no. 1; p. 273
• Main Authors: Kwon, Jiwoong, Park, Jong-Seok, Kang, Minsu, Choi, Soobin, Park, Jumi, Kim, Gyeong Tae, Lee, Changwook, Cha, Sangwon, Rhee, Hyun-Woo, Shim, Sang-Hee
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14-01-2020; Nature Publishing Group; Nature Portfolio
• Subjects: 14/35; 14/63; 38/109; 631/1647; 631/1647/2204; 631/1647/245; 631/1647/328; 631/57; 82/16; 82/58; Bilirubin; Bilirubin - metabolism; Binding; Covalent bonds; Dissolved oxygen; Fluorescence; Genetic code; Green Fluorescent Proteins - metabolism; Humanities and Social Sciences; Image resolution; Kinetics; Ligands; Light; Light intensity; Localization; Luminous intensity; Metabolites; Microscopy; Microscopy, Fluorescence; multidisciplinary; Oxidation; Photochemical Processes; Photooxidation; Protein Binding; Proteins; Reaction kinetics; Reaction mechanisms; Science; Science (multidisciplinary); Single Molecule Imaging - methods; Stability; Switching
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-14067-4

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.
Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin.