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Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Virgile Adam, Kyprianos Hadjidemetriou, Nickels Jensen, Robert L. Shoeman, Joyce Woodhouse, Andrew Aquila, Anne-Sophie Banneville, Thomas R. M. Barends, Victor Bezchastnov, Sébastien Boutet, Martin Byrdin, Marco Cammarata, Sergio Carbajo, Nina Eleni Christou, Nicolas Coquelle, Eugenio De la Mora, Mariam El Khatib, Tadeo Moreno Chicano, R. Bruce Doak, Franck Fieschi, Lutz Foucar, Oleksandr Glushonkov, Alexander Gorel, Marie Luise Grünbein, Mario Hilpert, Mark Hunter, Marco Kloos, Jason E. Koglin, Thomas J. Lane, Mengning Liang, Angela Mantovanelli, Karol Nass, Gabriela Nass Kovacs, Shigeki Owada, Christopher M. Roome, Giorgio Schirò, Matthew Seaberg, Miriam Stricker, Michel Thépaut, Kensuke Tono, Kiyoshi Ueda, Lucas M. Uriarte, Daehyun You, Ninon Zala, Tatiana Domratcheva, Stefan Jakobs, Michel Sliwa, Ilme Schlichting, Jacques-Philippe Colletier, Dominique Bourgeois, Martin Weik
doi: https://doi.org/10.1101/2021.11.05.462999
Virgile Adam
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Kyprianos Hadjidemetriou
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Nickels Jensen
2Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany and University Medical Center of Göttingen, Clinic for Neurology, Göttingen, Germany
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Robert L. Shoeman
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Joyce Woodhouse
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Andrew Aquila
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Anne-Sophie Banneville
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Thomas R. M. Barends
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Victor Bezchastnov
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Sébastien Boutet
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Martin Byrdin
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Marco Cammarata
5Department of Physics, UMR UR1-CNRS 6251, University of Rennes 1, Rennes, France
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Sergio Carbajo
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Nina Eleni Christou
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Nicolas Coquelle
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Eugenio De la Mora
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Mariam El Khatib
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Tadeo Moreno Chicano
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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R. Bruce Doak
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Franck Fieschi
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Lutz Foucar
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Oleksandr Glushonkov
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Alexander Gorel
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Marie Luise Grünbein
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Mario Hilpert
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Mark Hunter
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Marco Kloos
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Jason E. Koglin
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Thomas J. Lane
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Mengning Liang
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Angela Mantovanelli
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Karol Nass
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Gabriela Nass Kovacs
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Shigeki Owada
6RIKEN SPring-8 Center, Sayo, Japan
7Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan
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Christopher M. Roome
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Giorgio Schirò
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Matthew Seaberg
4Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA 94025, USA
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Miriam Stricker
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Michel Thépaut
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Kensuke Tono
6RIKEN SPring-8 Center, Sayo, Japan
7Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan
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Kiyoshi Ueda
8Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan
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Lucas M. Uriarte
9Univ. Lille, CNRS, UMR 8516, LASIR, Laboratoire de Spectroscopie pour les Interactions, la Réactivité et l’Environnement, Lille 59000, France
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Daehyun You
8Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan
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Ninon Zala
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Tatiana Domratcheva
10Department of Chemistry, Lomonosov Moscow State University, Moscow 119991, Russia
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  • For correspondence: Tatjana.Domratcheva@mpimf-heidelberg.mpg.de weik@ibs.fr
Stefan Jakobs
2Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany and University Medical Center of Göttingen, Clinic for Neurology, Göttingen, Germany
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Michel Sliwa
9Univ. Lille, CNRS, UMR 8516, LASIR, Laboratoire de Spectroscopie pour les Interactions, la Réactivité et l’Environnement, Lille 59000, France
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Ilme Schlichting
3Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120 Heidelberg, Germany
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Jacques-Philippe Colletier
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Dominique Bourgeois
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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Martin Weik
1Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044 Grenoble, France
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  • For correspondence: Tatjana.Domratcheva@mpimf-heidelberg.mpg.de weik@ibs.fr
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Abstract

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Competing Interest Statement

The authors have declared no competing interest.

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Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement
Virgile Adam, Kyprianos Hadjidemetriou, Nickels Jensen, Robert L. Shoeman, Joyce Woodhouse, Andrew Aquila, Anne-Sophie Banneville, Thomas R. M. Barends, Victor Bezchastnov, Sébastien Boutet, Martin Byrdin, Marco Cammarata, Sergio Carbajo, Nina Eleni Christou, Nicolas Coquelle, Eugenio De la Mora, Mariam El Khatib, Tadeo Moreno Chicano, R. Bruce Doak, Franck Fieschi, Lutz Foucar, Oleksandr Glushonkov, Alexander Gorel, Marie Luise Grünbein, Mario Hilpert, Mark Hunter, Marco Kloos, Jason E. Koglin, Thomas J. Lane, Mengning Liang, Angela Mantovanelli, Karol Nass, Gabriela Nass Kovacs, Shigeki Owada, Christopher M. Roome, Giorgio Schirò, Matthew Seaberg, Miriam Stricker, Michel Thépaut, Kensuke Tono, Kiyoshi Ueda, Lucas M. Uriarte, Daehyun You, Ninon Zala, Tatiana Domratcheva, Stefan Jakobs, Michel Sliwa, Ilme Schlichting, Jacques-Philippe Colletier, Dominique Bourgeois, Martin Weik
bioRxiv 2021.11.05.462999; doi: https://doi.org/10.1101/2021.11.05.462999
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Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement
Virgile Adam, Kyprianos Hadjidemetriou, Nickels Jensen, Robert L. Shoeman, Joyce Woodhouse, Andrew Aquila, Anne-Sophie Banneville, Thomas R. M. Barends, Victor Bezchastnov, Sébastien Boutet, Martin Byrdin, Marco Cammarata, Sergio Carbajo, Nina Eleni Christou, Nicolas Coquelle, Eugenio De la Mora, Mariam El Khatib, Tadeo Moreno Chicano, R. Bruce Doak, Franck Fieschi, Lutz Foucar, Oleksandr Glushonkov, Alexander Gorel, Marie Luise Grünbein, Mario Hilpert, Mark Hunter, Marco Kloos, Jason E. Koglin, Thomas J. Lane, Mengning Liang, Angela Mantovanelli, Karol Nass, Gabriela Nass Kovacs, Shigeki Owada, Christopher M. Roome, Giorgio Schirò, Matthew Seaberg, Miriam Stricker, Michel Thépaut, Kensuke Tono, Kiyoshi Ueda, Lucas M. Uriarte, Daehyun You, Ninon Zala, Tatiana Domratcheva, Stefan Jakobs, Michel Sliwa, Ilme Schlichting, Jacques-Philippe Colletier, Dominique Bourgeois, Martin Weik
bioRxiv 2021.11.05.462999; doi: https://doi.org/10.1101/2021.11.05.462999

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