Open Access
International Journal of Molecular Sciences, volume 22, issue 23, pages 12887
Lssmscarlet, dcyrfp2s, dcyofp2s and crispred2s, genetically encoded red fluorescent proteins with a large stokes shift
Subach Oksana M
1
,
Vlaskina Anna V
1
,
Agapova Yuliya K
1
,
Nikolaeva Alena Y.
1, 2
,
Ivashkina Olga I
1, 3, 4
,
Popov Vladimir O.
1, 2, 5
,
Piatkevich Kiryl D
6, 7, 8
,
Khrenova Maria G.
2, 9
,
Smirnova Tatiana A.
10
,
1
2
3
6
School of Life Sciences, Westlake University, Hangzhou 310024, China
|
7
Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou 310024, China
|
8
Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou 310024, China
|
Publication type: Journal Article
Publication date: 2021-11-28
Quartile SCImago
Q1
Quartile WOS
Q1
Impact factor: 5.6
ISSN: 16616596, 14220067
Catalysis
Organic Chemistry
Inorganic Chemistry
Physical and Theoretical Chemistry
Computer Science Applications
Spectroscopy
Molecular Biology
General Medicine
Abstract
Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.
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3 publications, 33.33%
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- We do not take into account publications that without a DOI.
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- Statistics recalculated weekly.
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Subach O. M. et al. Lssmscarlet, dcyrfp2s, dcyofp2s and crispred2s, genetically encoded red fluorescent proteins with a large stokes shift // International Journal of Molecular Sciences. 2021. Vol. 22. No. 23. p. 12887.
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Subach O. M., Vlaskina A. V., Agapova Y. K., Dorovatovskii P. V., Nikolaeva A. Y., Ivashkina O. I., Popov V. O., Piatkevich K. D., Khrenova M. G., Smirnova T. A., Boyko K. M., Subach F. V. Lssmscarlet, dcyrfp2s, dcyofp2s and crispred2s, genetically encoded red fluorescent proteins with a large stokes shift // International Journal of Molecular Sciences. 2021. Vol. 22. No. 23. p. 12887.
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TY - JOUR
DO - 10.3390/ijms222312887
UR - https://doi.org/10.3390%2Fijms222312887
TI - Lssmscarlet, dcyrfp2s, dcyofp2s and crispred2s, genetically encoded red fluorescent proteins with a large stokes shift
T2 - International Journal of Molecular Sciences
AU - Vlaskina, Anna V
AU - Agapova, Yuliya K
AU - Dorovatovskii, Pavel V.
AU - Nikolaeva, Alena Y.
AU - Ivashkina, Olga I
AU - Popov, Vladimir O.
AU - Subach, Oksana M
AU - Piatkevich, Kiryl D
AU - Khrenova, Maria G.
AU - Smirnova, Tatiana A.
AU - Boyko, Konstantin M.
AU - Subach, Fedor V.
PY - 2021
DA - 2021/11/28 00:00:00
PB - Multidisciplinary Digital Publishing Institute (MDPI)
SP - 12887
IS - 23
VL - 22
SN - 1661-6596
SN - 1422-0067
ER -
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@article{2021_Subach,
author = {Anna V Vlaskina and Yuliya K Agapova and Pavel V. Dorovatovskii and Alena Y. Nikolaeva and Olga I Ivashkina and Vladimir O. Popov and Oksana M Subach and Kiryl D Piatkevich and Maria G. Khrenova and Tatiana A. Smirnova and Konstantin M. Boyko and Fedor V. Subach},
title = {Lssmscarlet, dcyrfp2s, dcyofp2s and crispred2s, genetically encoded red fluorescent proteins with a large stokes shift},
journal = {International Journal of Molecular Sciences},
year = {2021},
volume = {22},
publisher = {Multidisciplinary Digital Publishing Institute (MDPI)},
month = {nov},
url = {https://doi.org/10.3390%2Fijms222312887},
number = {23},
pages = {12887},
doi = {10.3390/ijms222312887}
}
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Subach, Oksana M., et al. “Lssmscarlet, dcyrfp2s, dcyofp2s and crispred2s, genetically encoded red fluorescent proteins with a large stokes shift.” International Journal of Molecular Sciences, vol. 22, no. 23, Nov. 2021, p. 12887. https://doi.org/10.3390%2Fijms222312887.