The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.

Autor: Subach OM; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', 123182 Moscow, Russia., Tashkeev A; Unit of Animal Genomics, GIGA Research Center, University of Liège, 4000 Liege, Belgium., Vlaskina AV; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', 123182 Moscow, Russia., Petrenko DE; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', 123182 Moscow, Russia., Gaivoronskii FA; Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.; Faculty of Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia., Nikolaeva AY; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', 123182 Moscow, Russia.; Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia., Ivashkina OI; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', 123182 Moscow, Russia.; Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal Physiology, 125315 Moscow, Russia.; Institute for Advanced Brain Studies, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia., Anokhin KV; Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal Physiology, 125315 Moscow, Russia.; Institute for Advanced Brain Studies, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia., Popov VO; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', 123182 Moscow, Russia.; Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.; Faculty of Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia., Boyko KM; Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.; Moscow Institute of Physics and Technology, Institutsky Lane 9, Dolgoprudny, 141700 Moscow, Russia., Subach FV; Complex of NBICS Technologies, National Research Center 'Kurchatov Institute', 123182 Moscow, Russia.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2022 Mar 16; Vol. 23 (6). Date of Electronic Publication: 2022 Mar 16.
DOI: 10.3390/ijms23063208
Abstrakt: Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2-3- and 5-7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.
Databáze: MEDLINE
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