Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties
Abstract Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single‐particle‐t...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2024-03-01
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| Series: | Advanced Science |
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| Online Access: | https://doi.org/10.1002/advs.202306272 |
| _version_ | 1797263479256645632 |
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| author | Arijit Maity Jip Wulffelé Isabel Ayala Adrien Favier Virgile Adam Dominique Bourgeois Bernhard Brutscher |
| author_facet | Arijit Maity Jip Wulffelé Isabel Ayala Adrien Favier Virgile Adam Dominique Bourgeois Bernhard Brutscher |
| author_sort | Arijit Maity |
| collection | DOAJ |
| description | Abstract Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single‐particle‐tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side‐chain protonation of the conserved residues E212 and H62, altering the hydrogen‐bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green‐to‐Red photoconversion properties differ between the A and B states. The chromophore in the A‐state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B‐state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry. |
| first_indexed | 2024-04-25T00:13:39Z |
| format | Article |
| id | doaj.art-a2ba798876974fd1b3d51ee2afb49cd0 |
| institution | Directory Open Access Journal |
| issn | 2198-3844 |
| language | English |
| last_indexed | 2024-04-25T00:13:39Z |
| publishDate | 2024-03-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj.art-a2ba798876974fd1b3d51ee2afb49cd02024-03-13T07:30:35ZengWileyAdvanced Science2198-38442024-03-011110n/an/a10.1002/advs.202306272Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical PropertiesArijit Maity0Jip Wulffelé1Isabel Ayala2Adrien Favier3Virgile Adam4Dominique Bourgeois5Bernhard Brutscher6CEA CNRS Institut de Biologie Structurale (IBS) Université Grenoble Alpes 71 avenue des Martyrs, Cedex 9 Grenoble 38044 FranceCEA CNRS Institut de Biologie Structurale (IBS) Université Grenoble Alpes 71 avenue des Martyrs, Cedex 9 Grenoble 38044 FranceCEA CNRS Institut de Biologie Structurale (IBS) Université Grenoble Alpes 71 avenue des Martyrs, Cedex 9 Grenoble 38044 FranceCEA CNRS Institut de Biologie Structurale (IBS) Université Grenoble Alpes 71 avenue des Martyrs, Cedex 9 Grenoble 38044 FranceCEA CNRS Institut de Biologie Structurale (IBS) Université Grenoble Alpes 71 avenue des Martyrs, Cedex 9 Grenoble 38044 FranceCEA CNRS Institut de Biologie Structurale (IBS) Université Grenoble Alpes 71 avenue des Martyrs, Cedex 9 Grenoble 38044 FranceCEA CNRS Institut de Biologie Structurale (IBS) Université Grenoble Alpes 71 avenue des Martyrs, Cedex 9 Grenoble 38044 FranceAbstract Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single‐particle‐tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side‐chain protonation of the conserved residues E212 and H62, altering the hydrogen‐bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green‐to‐Red photoconversion properties differ between the A and B states. The chromophore in the A‐state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B‐state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.https://doi.org/10.1002/advs.202306272fluorescencePCFPproteinsolution NMRsuper‐resolution microscopy |
| spellingShingle | Arijit Maity Jip Wulffelé Isabel Ayala Adrien Favier Virgile Adam Dominique Bourgeois Bernhard Brutscher Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties Advanced Science fluorescence PCFP protein solution NMR super‐resolution microscopy |
| title | Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties |
| title_full | Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties |
| title_fullStr | Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties |
| title_full_unstemmed | Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties |
| title_short | Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties |
| title_sort | structural heterogeneity in a phototransformable fluorescent protein impacts its photochemical properties |
| topic | fluorescence PCFP protein solution NMR super‐resolution microscopy |
| url | https://doi.org/10.1002/advs.202306272 |
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