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The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer
Oksana M Subach1, Aleksandr Tashkeev2, Anna V Vlaskina1
1Complex of NBICS Technologies, National Research Center "Kurchatov Institute", 123182 Moscow, Russia.
Abstract:
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.
Insights
Researchers developed mRubyFT, a brighter blue-to-red fluorescent timer, overcoming limitations of earlier protein timers. This new tool enhances protein tracking and visualization in live cells, offering improved brightness for biological research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) are valuable for tracking protein age, trafficking, and cell labeling.
- Existing mCherry-derived mFTs exhibit reduced brightness compared to enhanced green fluorescent protein (EGFP).
Purpose of the Study:
- To develop a brighter blue-to-red fluorescent timer to overcome the brightness limitations of existing mFTs.
- To characterize the properties and applications of the novel mRubyFT timer.
Main Methods:
- Development of mRubyFT derived from mRuby2 red fluorescent protein.
- In vitro and in vivo characterization of mRubyFT brightness, maturation time, and photoconversion efficiency.
- Confocal imaging of cytoskeleton proteins in live mammalian cells.
- X-ray crystallography and site-directed mutagenesis to analyze the mRubyFT structure.
Main Results:
- mRubyFT exhibits significantly improved brightness in both blue and red forms compared to mCherry-derived Fast-FT.
- mRubyFT demonstrates a maturation half-time of 15 hours and maximum blue form at 5.7 hours.
- Photoconversion efficiency of mRubyFT is lower than Fast-FT, but its monomeric nature facilitates cytoskeleton labeling.
- X-ray structure analysis revealed insights into chromophore-surrounding residues.
Conclusions:
- mRubyFT represents a significant advancement in fluorescent timer technology, offering enhanced brightness for biological applications.
- The improved brightness and monomeric properties of mRubyFT expand its utility in live-cell imaging and protein dynamics studies.
- Structural analysis provides a foundation for further optimization of fluorescent timer proteins.
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