Title :

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Author(s) :

Adam, Virgile [Auteur]
Institut de biologie structurale [IBS - UMR 5075]
Hadjidemetriou, K. [Auteur]
Jensen, N. [Auteur]
Shoeman, R. L. [Auteur]
Woodhouse, J. [Auteur]
Aquila, A. [Auteur]
Banneville, A. S. [Auteur]
Barends, T. R. M. [Auteur]
Bezchastnov, V. [Auteur]
Boutet, S. [Auteur]
Byrdin, M. [Auteur]
Cammarata, Marco [Auteur]
Institut de Physique de Rennes [IPR]
Carbajo, S. [Auteur]
Eleni Christou, N. [Auteur]
Coquelle, N. [Auteur]
De La Mora, E. [Auteur]
El Khatib, M. [Auteur]
Moreno Chicano, T. [Auteur]
Bruce Doak, R. [Auteur]
Fieschi, F. [Auteur]
Foucar, L. [Auteur]
Glushonkov, O. [Auteur]
Gorel, A. [Auteur]
Grünbein, M. L. [Auteur]
Hilpert, M. [Auteur]
Hunter, M. [Auteur]
Kloos, M. [Auteur]
Koglin, J. E. [Auteur]
Lane, T. J. [Auteur]
Liang, M. [Auteur]
Mantovanelli, A. [Auteur]
Nass, K. [Auteur]
Nass Kovacs, G. [Auteur]
Owada, S. [Auteur]
Roome, C. M. [Auteur]
Schirò, G. [Auteur]
Seaberg, M. [Auteur]
Stricker, M. [Auteur]
Thépaut, M. [Auteur]
Tono, K. [Auteur]
Ueda, K. [Auteur]
Uriarte, L. M. [Auteur]
You, D. [Auteur]
Zala, N. [Auteur]
Domratcheva, T. [Auteur]
Jakobs, S. [Auteur]
Sliwa, Michel [Auteur]
Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement (LASIRE) - UMR 8516
Schlichting, I. [Auteur]
Colletier, J. P. [Auteur]
Bourgeois, D. [Auteur]
Weik, M. [Auteur]

Journal title :

ChemPhysChem

Abbreviated title :

Chemphyschem

Pages :

e202200192

Publication date :

2022-08-14

ISSN :

1439-7641

English keyword(s) :

quantum chemistry
switching contrast
serial femtosecond crystallography
photoswitchable fluorescent proteins
nanoscopy

HAL domain(s) :

Sciences du Vivant [q-bio]/Biochimie, Biologie Moléculaire/Biologie structurale [q-bio.BM]
Physique [physics]

English abstract : [en]

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 ...
Show more >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-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.Show less >

Language :

Anglais

Peer reviewed article :

Oui

Audience :

Internationale

Popular science :

Non

Administrative institution(s) :

Université de Lille
CNRS

Collections :

• Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement (LASIRE) - UMR 8516

Submission date :

2024-02-28T22:21:36Z
2024-03-19T09:49:25Z