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

Jiwoong Kwon, Jong-Seok Park, Minsu Kang, Soobin Choi, Jumi Park, Gyeong Tae Kim, Changwook Lee, Sangwon Cha, Hyun-Woo Rhee () and Sang-Hee Shim ()
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Jiwoong Kwon: Institute for Basic Science (IBS)
Jong-Seok Park: Ulsan National Institute of Science and Technology (UNIST)
Minsu Kang: Institute for Basic Science (IBS)
Soobin Choi: Hankuk University of Foreign Studies
Jumi Park: Ulsan National Institute of Science and Technology (UNIST)
Gyeong Tae Kim: Ulsan National Institute of Science and Technology (UNIST)
Changwook Lee: Ulsan National Institute of Science and Technology (UNIST)
Sangwon Cha: Hankuk University of Foreign Studies
Hyun-Woo Rhee: Ulsan National Institute of Science and Technology (UNIST)
Sang-Hee Shim: Institute for Basic Science (IBS)

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: Abstract 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.

Date: 2020
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DOI: 10.1038/s41467-019-14067-4

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