Rational control of off‐state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement
Virgile Adam
(1)
,
Kyprianos Hadjidemetriou
(1)
,
Nickels Jensen
(2)
,
Robert L. Shoeman
(3)
,
Joyce Woodhouse
(1)
,
Andrew Aquila
(4)
,
Anne-Sophie Banneville
(1)
,
Thomas Barends
(5)
,
Victor Bezchastnov
(5)
,
Sébastien Boutet
(4)
,
Martin Byrdin
(1)
,
Marco Cammarata
(6)
,
Sergio Carbajo
(4)
,
Nina Eleni Christou
(1)
,
Nicolas Coquelle
(1)
,
Eugenio de la Mora
(1)
,
Mariam El Khatib
(1)
,
Tadeo Moreno Chicano
(1)
,
R. Bruce Doak
(5)
,
Franck Fieschi
(1)
,
Lutz Foucar
(5)
,
Oleksandr Glushonkov
(1)
,
Alexander Gorel
(5)
,
Marie Luise Grünbein
(5)
,
Mario Hilpert
(5)
,
Mark Hunter
(4)
,
Marco Kloos
(5)
,
Jason Koglin
(4)
,
Thomas J. Lane
(4)
,
Mengning Liang
(4)
,
Angela Mantovanelli
(1)
,
Karol Nass
(5)
,
Gabriela Nass Kovacs
(5)
,
Shigeki Owada
(7, 8)
,
Christopher M. Roome
(5)
,
Giorgio Schirò
(1)
,
Matthew Seaberg
(4)
,
Miriam Stricker
(5)
,
Michel Thépaut
(1)
,
Kensuke Tono
(7, 8)
,
Kiyoshi Ueda
(9)
,
Lucas M. Uriarte
(10)
,
Daehyun You
(9)
,
Ninon Zala
(1)
,
Tatiana Domratcheva
(5, 11)
,
Stefan Jakobs
(2)
,
Michel Sliwa
(10)
,
Ilme Schlichting
(5)
,
Jacques-Philippe Colletier
(1)
,
Dominique Bourgeois
(1)
,
Martin Weik
(1)
1
IBS - UMR 5075 -
Institut de biologie structurale
2 Department of NanoBiophotonics [Göttingen]
3 Max Planck Institute for Medical Research [Heidelberg]
4 LCLS - Linac Coherent Light Source
5 Max-Planck-Institut für Medizinische Forschung
6 IPR - Institut de Physique de Rennes
7 RIKEN RSC - RIKEN SPring-8 Center [Hyogo]
8 JASRI - Japan Synchrotron Radiation Research Institute [Hyogo]
9 Tohoku University [Sendai]
10 LASIRE - Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516
11 Department of Chemistry, Lomonosov Moscow State University
2 Department of NanoBiophotonics [Göttingen]
3 Max Planck Institute for Medical Research [Heidelberg]
4 LCLS - Linac Coherent Light Source
5 Max-Planck-Institut für Medizinische Forschung
6 IPR - Institut de Physique de Rennes
7 RIKEN RSC - RIKEN SPring-8 Center [Hyogo]
8 JASRI - Japan Synchrotron Radiation Research Institute [Hyogo]
9 Tohoku University [Sendai]
10 LASIRE - Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516
11 Department of Chemistry, Lomonosov Moscow State University
Virgile Adam
- Fonction : Auteur
- PersonId : 4048
- IdHAL : virgile-adam
- ORCID : 0000-0003-2209-7846
- IdRef : 250406187
Kyprianos Hadjidemetriou
- Fonction : Auteur
- PersonId : 1167494
- ORCID : 0000-0003-1918-763X
Anne-Sophie Banneville
- Fonction : Auteur
- PersonId : 1184746
- ORCID : 0000-0002-0698-6291
Thomas Barends
- Fonction : Auteur
- PersonId : 1167495
- ORCID : 0000-0002-9488-3005
Sébastien Boutet
- Fonction : Auteur
- PersonId : 1184759
- ORCID : 0000-0003-3928-1244
Martin Byrdin
- Fonction : Auteur
- PersonId : 1638
- IdHAL : martin-byrdin
- ORCID : 0000-0002-6389-8714
- IdRef : 185869890
Marco Cammarata
- Fonction : Auteur
- PersonId : 491
- IdHAL : marco-cammarata
- ORCID : 0000-0003-3013-1186
- IdRef : 229610080
Franck Fieschi
- Fonction : Auteur
- PersonId : 761070
- ORCID : 0000-0003-1194-8107
- IdRef : 087253240
Mark Hunter
- Fonction : Auteur
- PersonId : 1184748
- ORCID : 0000-0002-0110-7075
Karol Nass
- Fonction : Auteur
- PersonId : 773540
- ORCID : 0000-0001-8843-1566
Shigeki Owada
- Fonction : Auteur
- PersonId : 780427
- ORCID : 0000-0002-1451-7612
Giorgio Schirò
- Fonction : Auteur
- PersonId : 742723
- IdHAL : giorgio-schiro
Michel Thépaut
- Fonction : Auteur
- PersonId : 802375
- ORCID : 0000-0003-4792-8271
Kensuke Tono
- Fonction : Auteur
- PersonId : 780426
- ORCID : 0000-0003-1218-3759
Lucas M. Uriarte
- Fonction : Auteur
- PersonId : 1133097
Michel Sliwa
- Fonction : Auteur
- PersonId : 17467
- IdHAL : michel-sliwa
- ORCID : 0000-0002-5073-8180
- IdRef : 08084166X
Ilme Schlichting
- Fonction : Auteur
- PersonId : 1167496
- ORCID : 0000-0002-0936-7496
Jacques-Philippe Colletier
- Fonction : Auteur
- PersonId : 739662
- IdHAL : jacques-philippe-colletier
- ORCID : 0000-0003-1819-4686
- IdRef : 110264983
Dominique Bourgeois
- Fonction : Auteur
- PersonId : 1634
- IdHAL : dominique-bourgeois
- ORCID : 0000-0002-1862-7712
- IdRef : 079965415
Martin Weik
- Fonction : Auteur
- PersonId : 1157762
- ORCID : 0000-0001-9297-642X
- IdRef : 11026486X
Résumé
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, one of the key parameters that dictate 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-leve l 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.
| Origine | Accord explicite pour ce dépôt |
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