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

Jiwoong Kwon,Changwook Lee,Soobin Choi,Gyeong Tae Kim,Hyun-Woo Rhee,Jumi Park,Minsu Kang,Sangwon Cha,Jong-Seok Park,Sang-Hee Shim

doi:10.1038/s41467-019-14067-4

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.

Highlights

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of highquality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein
We performed Single-molecule localization (SML) imaging of various subcellular structures at sub-diffractionlimited resolutions, utilizing the advantages of UnaG such as genetic incorporation for labeling, background-free imaging under Epi illumination, controllable UV-free switching kinetics ensuring both of high contrast ratio and fast acquisition, and crosstalk-free multicolor imaging of live cells
In GFP and photoswitching proteins used in SML imaging, the chromophores are formed from a series of reactions between the amino acids of the proteins (Supplementary Fig. 1a, d, e)

Summary

Introduction

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of highquality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. A protein of interest (POI) in a live cell can be labeled with an organic dye via self-labeling enzymes such as FlAsH, SNAP, CLIP and Halo[16,17,18,19] These labeling schemes suffer from high background fluorescence from non-specific binding of organic dyes to the substrate and cellular membranes. The application area of UnaG was further extended to monitor the activity of membrane transporters[34], to regulate the activity of a POI35, to report the protein–protein interactions[36], to investigate hypoxia states of cells[37,38], and to monitor calcium and BR simultaneously[39] Despite these various applications as sensors, UnaG has not been extensively used in general imaging applications probably due to the sensitive photobleaching through BR photo-oxidation, which we characterized and utilized in this article

Methods

Results

Conclusion