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Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants

The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening t...

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Autores principales: Mamontova, Anastasia V., Shakhov, Aleksander M., Lukyanov, Konstantin A., Bogdanov, Alexey M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696298/
https://www.ncbi.nlm.nih.gov/pubmed/33202759
http://dx.doi.org/10.3390/biom10111547
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author Mamontova, Anastasia V.
Shakhov, Aleksander M.
Lukyanov, Konstantin A.
Bogdanov, Alexey M.
author_facet Mamontova, Anastasia V.
Shakhov, Aleksander M.
Lukyanov, Konstantin A.
Bogdanov, Alexey M.
author_sort Mamontova, Anastasia V.
collection PubMed
description The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear. In this work, we show that the Y145M substitution, both alone and in combination with the F165Y mutation, does not shorten the fluorescence lifetime of EGFP-derived mutants. Thus, the unlocking of Y145M as an important determinant of lifetime tuning is possible only cooperatively with mutations at position 65. We also show here that the introduction of a T65G substitution into EGFP causes complex photobehavior of the respective mutants in the lifetime domain, namely, the appearance of two fluorescent states with different lifetimes, preserved in any combination with the Y145M and F165Y substitutions.
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spelling pubmed-76962982020-11-29 Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants Mamontova, Anastasia V. Shakhov, Aleksander M. Lukyanov, Konstantin A. Bogdanov, Alexey M. Biomolecules Article The bright ultimately short lifetime enhanced emitter (BrUSLEE) green fluorescent protein, which differs from the enhanced green fluorescent protein (EGFP) in three mutations, exhibits an extremely short fluorescence lifetime at a relatively high brightness. An important contribution to shortening the BrUSLEE fluorescence lifetime compared to EGFP is provided by the T65G substitution of chromophore-forming residue and the Y145M mutation touching the chromophore environment. Although the influence of the T65G mutation was studied previously, the role of the 145th position in determining the GFPs physicochemical characteristics remains unclear. In this work, we show that the Y145M substitution, both alone and in combination with the F165Y mutation, does not shorten the fluorescence lifetime of EGFP-derived mutants. Thus, the unlocking of Y145M as an important determinant of lifetime tuning is possible only cooperatively with mutations at position 65. We also show here that the introduction of a T65G substitution into EGFP causes complex photobehavior of the respective mutants in the lifetime domain, namely, the appearance of two fluorescent states with different lifetimes, preserved in any combination with the Y145M and F165Y substitutions. MDPI 2020-11-13 /pmc/articles/PMC7696298/ /pubmed/33202759 http://dx.doi.org/10.3390/biom10111547 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Mamontova, Anastasia V.
Shakhov, Aleksander M.
Lukyanov, Konstantin A.
Bogdanov, Alexey M.
Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants
title Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants
title_full Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants
title_fullStr Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants
title_full_unstemmed Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants
title_short Deciphering the Role of Positions 145 and 165 in Fluorescence Lifetime Shortening in the EGFP Variants
title_sort deciphering the role of positions 145 and 165 in fluorescence lifetime shortening in the egfp variants
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696298/
https://www.ncbi.nlm.nih.gov/pubmed/33202759
http://dx.doi.org/10.3390/biom10111547
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