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13 May 2022
Article (Scientific journals)
The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.
Subach, Oksana M; Tashkeev, Aleksandr  ; Vlaskina, Anna V et al.
2022 • In International Journal of Molecular Sciences, 23 (6), p. 3208
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Keywords :
RubyFT#11-9; X-ray structure; fluorescence imaging; fluorescent protein; genetically encoded blue-to-red fluorescent timers; mRubyFT; protein engineering; Luminescent Proteins; Green Fluorescent Proteins; Animals; Green Fluorescent Proteins/chemistry; Green Fluorescent Proteins/genetics; Luminescent Proteins/metabolism; Mutagenesis, Site-Directed; Light; Mammals/metabolism; Catalysis; Molecular Biology; Spectroscopy; Computer Science Applications; Physical and Theoretical Chemistry; Organic Chemistry; Inorganic Chemistry; General Medicine
Abstract :
[en] 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.
Disciplines :
Biotechnology
Author, co-author :
Subach, Oksana M;  Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia
Tashkeev, Aleksandr ;  Université de Liège - ULiège > GIGA > GIGA Medical Genomics - Unit of Animal Genomics
Vlaskina, Anna V;  Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia
Petrenko, Dmitry E;  Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia
Gaivoronskii, Filipp A;  Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia
Nikolaeva, Alena Y;  Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia
Ivashkina, Olga I;  Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia
Anokhin, Konstantin V;  Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal Physiology, 125315 Moscow, Russia
Popov, Vladimir O;  Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia
Boyko, Konstantin M ;  Bach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia
Subach, Fedor V ;  Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia
Language :
English
Title :
The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.
Publication date :
16 March 2022
Journal title :
International Journal of Molecular Sciences
ISSN :
1661-6596
eISSN :
1422-0067
Publisher :
MDPI, Switzerland
Volume :
23
Issue :
6
Pages :
3208
Peer reviewed :
Peer Reviewed verified by ORBi
Funders :
NRC Kurchatov institute
Russian Foundation for Basic Research
Russian Science Foundation
Funding text :
Funding: This research was funded by the NRC Kurchatov Institute and an internal grant of the National Research Center, Kurchatov Institute No 2752 of 28.10.2021 (developing the mRubyFT timer); by Fonds de la Recherche Scientifique de Belgique (F.R.S.-FNRS) under ASP/AR 414 grant to A.T. (development of RubyFT#11-9); by RFBR grant No 19-04-00395 to O.M.S. (directed mutagenesis of mRubyFT); by the Russian Science Foundation grant No 21-74-20135 to K.M.B. (crystallization, data collection, and structural studies); by RFBR grant No 20-015-00427 to O.I.I. (characterization of the blue-to-red photoconversion); and by the Ministry of Science and Higher Education of the Russian Federation grant No 075-15-2020-801 to K.V.A. (confocal imaging). The work was also supported by the Resource Centers department of the National Research Center, Kurchatov Institute (imaging of bacterial cells and cultivation of mammalian cells).

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