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Structural and photophysical characterization of the small ultra-red fluorescent protein

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further...

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Autores principales: Maiti, Atanu, Buffalo, Cosmo Z., Saurabh, Saumya, Montecinos-Franjola, Felipe, Hachey, Justin S., Conlon, William J., Tran, Geraldine N., Hassan, Bakar, Walters, Kylie J., Drobizhev, Mikhail, Moerner, W. E., Ghosh, Partho, Matsuo, Hiroshi, Tsien, Roger Y., Lin, John Y., Rodriguez, Erik A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10338489/
https://www.ncbi.nlm.nih.gov/pubmed/37438348
http://dx.doi.org/10.1038/s41467-023-39776-9
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author Maiti, Atanu
Buffalo, Cosmo Z.
Saurabh, Saumya
Montecinos-Franjola, Felipe
Hachey, Justin S.
Conlon, William J.
Tran, Geraldine N.
Hassan, Bakar
Walters, Kylie J.
Drobizhev, Mikhail
Moerner, W. E.
Ghosh, Partho
Matsuo, Hiroshi
Tsien, Roger Y.
Lin, John Y.
Rodriguez, Erik A.
author_facet Maiti, Atanu
Buffalo, Cosmo Z.
Saurabh, Saumya
Montecinos-Franjola, Felipe
Hachey, Justin S.
Conlon, William J.
Tran, Geraldine N.
Hassan, Bakar
Walters, Kylie J.
Drobizhev, Mikhail
Moerner, W. E.
Ghosh, Partho
Matsuo, Hiroshi
Tsien, Roger Y.
Lin, John Y.
Rodriguez, Erik A.
author_sort Maiti, Atanu
collection PubMed
description The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.
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spelling pubmed-103384892023-07-14 Structural and photophysical characterization of the small ultra-red fluorescent protein Maiti, Atanu Buffalo, Cosmo Z. Saurabh, Saumya Montecinos-Franjola, Felipe Hachey, Justin S. Conlon, William J. Tran, Geraldine N. Hassan, Bakar Walters, Kylie J. Drobizhev, Mikhail Moerner, W. E. Ghosh, Partho Matsuo, Hiroshi Tsien, Roger Y. Lin, John Y. Rodriguez, Erik A. Nat Commun Article The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies. Nature Publishing Group UK 2023-07-12 /pmc/articles/PMC10338489/ /pubmed/37438348 http://dx.doi.org/10.1038/s41467-023-39776-9 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Maiti, Atanu
Buffalo, Cosmo Z.
Saurabh, Saumya
Montecinos-Franjola, Felipe
Hachey, Justin S.
Conlon, William J.
Tran, Geraldine N.
Hassan, Bakar
Walters, Kylie J.
Drobizhev, Mikhail
Moerner, W. E.
Ghosh, Partho
Matsuo, Hiroshi
Tsien, Roger Y.
Lin, John Y.
Rodriguez, Erik A.
Structural and photophysical characterization of the small ultra-red fluorescent protein
title Structural and photophysical characterization of the small ultra-red fluorescent protein
title_full Structural and photophysical characterization of the small ultra-red fluorescent protein
title_fullStr Structural and photophysical characterization of the small ultra-red fluorescent protein
title_full_unstemmed Structural and photophysical characterization of the small ultra-red fluorescent protein
title_short Structural and photophysical characterization of the small ultra-red fluorescent protein
title_sort structural and photophysical characterization of the small ultra-red fluorescent protein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10338489/
https://www.ncbi.nlm.nih.gov/pubmed/37438348
http://dx.doi.org/10.1038/s41467-023-39776-9
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