The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer
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...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-03-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/6/3208 |
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| author | Oksana M. Subach Aleksandr Tashkeev Anna V. Vlaskina Dmitry E. Petrenko Filipp A. Gaivoronskii Alena Y. Nikolaeva Olga I. Ivashkina Konstantin V. Anokhin Vladimir O. Popov Konstantin M. Boyko Fedor V. Subach |
| author_facet | Oksana M. Subach Aleksandr Tashkeev Anna V. Vlaskina Dmitry E. Petrenko Filipp A. Gaivoronskii Alena Y. Nikolaeva Olga I. Ivashkina Konstantin V. Anokhin Vladimir O. Popov Konstantin M. Boyko Fedor V. Subach |
| author_sort | Oksana M. Subach |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T19:42:02Z |
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| id | doaj.art-073d6b6fdcc14757bf21b8c167ae74e7 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T19:42:02Z |
| publishDate | 2022-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-073d6b6fdcc14757bf21b8c167ae74e72023-11-24T01:34:51ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-03-01236320810.3390/ijms23063208The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent TimerOksana M. Subach0Aleksandr Tashkeev1Anna V. Vlaskina2Dmitry E. Petrenko3Filipp A. Gaivoronskii4Alena Y. Nikolaeva5Olga I. Ivashkina6Konstantin V. Anokhin7Vladimir O. Popov8Konstantin M. Boyko9Fedor V. Subach10Complex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaUnit of Animal Genomics, GIGA Research Center, University of Liège, 4000 Liege, BelgiumComplex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaComplex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaBach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, RussiaComplex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaComplex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaLaboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal Physiology, 125315 Moscow, RussiaComplex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaBach Institute of Biochemistry, Research Centre of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, RussiaComplex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, RussiaGenetically 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.https://www.mdpi.com/1422-0067/23/6/3208genetically encoded blue-to-red fluorescent timersprotein engineeringfluorescence imagingfluorescent proteinmRubyFTRubyFT#11-9 |
| spellingShingle | Oksana M. Subach Aleksandr Tashkeev Anna V. Vlaskina Dmitry E. Petrenko Filipp A. Gaivoronskii Alena Y. Nikolaeva Olga I. Ivashkina Konstantin V. Anokhin Vladimir O. Popov Konstantin M. Boyko Fedor V. Subach The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer International Journal of Molecular Sciences genetically encoded blue-to-red fluorescent timers protein engineering fluorescence imaging fluorescent protein mRubyFT RubyFT#11-9 |
| title | The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer |
| title_full | The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer |
| title_fullStr | The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer |
| title_full_unstemmed | The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer |
| title_short | The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer |
| title_sort | mrubyft protein genetically encoded blue to red fluorescent timer |
| topic | genetically encoded blue-to-red fluorescent timers protein engineering fluorescence imaging fluorescent protein mRubyFT RubyFT#11-9 |
| url | https://www.mdpi.com/1422-0067/23/6/3208 |
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