Structural and photophysical characterization of the small ultra-red fluorescent protein
Abstract 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 enabl...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-07-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-39776-9 |
| _version_ | 1797778790675382272 |
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| author | Atanu Maiti Cosmo Z. Buffalo Saumya Saurabh Felipe Montecinos-Franjola Justin S. Hachey William J. Conlon Geraldine N. Tran Bakar Hassan Kylie J. Walters Mikhail Drobizhev W. E. Moerner Partho Ghosh Hiroshi Matsuo Roger Y. Tsien John Y. Lin Erik A. Rodriguez |
| author_facet | Atanu Maiti Cosmo Z. Buffalo Saumya Saurabh Felipe Montecinos-Franjola Justin S. Hachey William J. Conlon Geraldine N. Tran Bakar Hassan Kylie J. Walters Mikhail Drobizhev W. E. Moerner Partho Ghosh Hiroshi Matsuo Roger Y. Tsien John Y. Lin Erik A. Rodriguez |
| author_sort | Atanu Maiti |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-12T23:22:39Z |
| format | Article |
| id | doaj.art-c7b97c5ea32a4f34823ce6ae4a9c9f7e |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-12T23:22:39Z |
| publishDate | 2023-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-c7b97c5ea32a4f34823ce6ae4a9c9f7e2023-07-16T11:22:03ZengNature PortfolioNature Communications2041-17232023-07-0114111410.1038/s41467-023-39776-9Structural and photophysical characterization of the small ultra-red fluorescent proteinAtanu Maiti0Cosmo Z. Buffalo1Saumya Saurabh2Felipe Montecinos-Franjola3Justin S. Hachey4William J. Conlon5Geraldine N. Tran6Bakar Hassan7Kylie J. Walters8Mikhail Drobizhev9W. E. Moerner10Partho Ghosh11Hiroshi Matsuo12Roger Y. Tsien13John Y. Lin14Erik A. Rodriguez15Cancer Innovation Laboratory, Frederick National Laboratory for Cancer ResearchDepartment of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, BerkeleyDepartment of Chemistry, Stanford UniversityDepartment of Chemistry, The George Washington UniversityDepartment of Chemistry, The George Washington UniversityDepartment of Chemistry, The George Washington UniversityDepartment of Radiology, Boston UniversityProtein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of HealthProtein Processing Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, National Institutes of HealthDepartment of Microbiology and Cell Biology, Montana State UniversityDepartment of Chemistry, Stanford UniversityDepartment of Chemistry and Biochemistry, University of California, San DiegoCancer Innovation Laboratory, Frederick National Laboratory for Cancer ResearchDepartment of Chemistry and Biochemistry, University of California, San DiegoTasmanian School of Medicine, University of TasmaniaDepartment of Chemistry, The George Washington UniversityAbstract 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.https://doi.org/10.1038/s41467-023-39776-9 |
| spellingShingle | Atanu Maiti Cosmo Z. Buffalo Saumya Saurabh Felipe Montecinos-Franjola Justin S. Hachey William J. Conlon Geraldine N. Tran Bakar Hassan Kylie J. Walters Mikhail Drobizhev W. E. Moerner Partho Ghosh Hiroshi Matsuo Roger Y. Tsien John Y. Lin Erik A. Rodriguez Structural and photophysical characterization of the small ultra-red fluorescent protein Nature Communications |
| 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 |
| url | https://doi.org/10.1038/s41467-023-39776-9 |
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