mEos3.2 is a photoconvertible fluorescence protein with comparatively low brightness, which limits its application in live Super resolution microscopy. To address this issue, we have used semi-rational protein engineering to develop mEosBrite, a new class of improved brightness variants. The improvement in the brightness was confirmed by expression in E.coli as well as mammalian cell lines. Furthermore, biophysical characterization suggests that all the three mEosBrite variant proteins display higher quantum yield, truly monomeric form, less cytotoxicity and lower protein aggregation as compared to the wild type mEos3.2 protein. Most importantly, because of their high photoconversion efficiency mEosBrite variants could be an excellent tool for single-molecule and intensity fluctuation based super-resolution microscopy.
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Photoconvertible Fluorescent Protein
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We thank Bhattacharyya lab members for critical review of the manuscripts and providing valuable input. The research was funded by the DBT research grant (No. BT/PR8072/MED/32/295/2013). PM. was funded by the ACTREC doctoral fellowship. DN thanks DBT-IISc program.
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Electronic supplementary material
Sequence alignment of mEos3.2 with mClavGR2. A) Sequence alignment of mClavGR1 and mClavGR2. Amino acid substitutions in mClavGR1 which led to generation of brighter variant mClavGR2 are highlighted. B) Sequence alignment of mEos3.2 and mClavGR2. The highlighted residues of mEos3.2 were substituted with the respective mClavGR2 residues in different combinations to get brighter variants. (PDF 296 kb)
SDS-PAGE of mEosBrite variants. His6-tagged mEosBrite variant proteins were purified using nickel affinity column method and analysed by SDS-PAGE (15%). All the purified protein variants gave a band just above 25KDa. (PDF 71 kb)
Spectral properties and maturation kinetics of mEosBrite variants. A) Excitation and Emission fluorescence spectra for green and red states of WT mEos3.2 and the mEosBrite variants. B) Absorbance spectra and C) Fluorescence maturation profile of WT mEos3.2 and the mEosBrite variants performed at 37°C using fluorescent plate reader. The maturation profiles of all proteins indicate maturation time is between 20-23 min. (PDF 151 kb)
pH dependent behaviour of mEosBrite variants. Fluorescence intensity of green and red states of WT and the mEosBrite variants was plotted against pH (2 to 12). Fluorescence intensities were measured using fluorescence plate reader. (PDF 95 kb)
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Marathe, P., H.S., M.S., Nair, D. et al. mEosBrite Are Bright Variants of mEos3.2 Developed by Semirational Protein Engineering. J Fluoresc 30, 703–715 (2020). https://doi.org/10.1007/s10895-020-02537-8
- Photoconvertible fluorescent protein
- Directed evolution
- Super-resolution microscopy