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Sci Rep. 2018 Sep 5;8(1):13224. doi: 10.1038/s41598-018-31687-w.

Bright GFP with subnanosecond fluorescence lifetime.

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Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia.
Semenov Institute of Chemical Physics, Moscow, Russia.
Moscow Institute of Physics and Technology, Dolgoprudny, Moscow region, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.


Fluorescence lifetime imaging microscopy (FLIM) measures fluorescence decay rate at every pixel of an image. FLIM can separate probes of the same color but different fluorescence lifetimes (FL), thus it is a promising approach for multiparameter imaging. However, available GFP-like fluorescent proteins (FP) possess a narrow range of FLs (commonly, 2.3-3.5 ns) which limits their applicability for multiparameter FLIM. Here we report a new FP probe showing both subnanosecond fluorescence lifetime and exceptional fluorescence brightness (80% of EGFP). To design this probe we applied semi-rational amino acid substitutions selection. Critical positions (Thr65, Tyr145, Phe165) were altered based on previously reported effect on FL or excited state electron transfer. The resulting EGFP triple mutant, BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), allows for both reliable detection of the probe and recording FL signal clearly distinguishable from that of the spectrally similar commonly used GFPs. We demonstrated high performance of this probe in multiparameter FLIM experiment. We suggest that amino acid substitutions described here lead to a significant shift in radiative and non-radiative excited state processes equilibrium.

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