PT - JOURNAL ARTICLE AU - Virgile Adam AU - Kyprianos Hadjidemetriou AU - Nickels Jensen AU - Robert L. Shoeman AU - Joyce Woodhouse AU - Andrew Aquila AU - Anne-Sophie Banneville AU - Thomas R. M. Barends AU - Victor Bezchastnov AU - Sébastien Boutet AU - Martin Byrdin AU - Marco Cammarata AU - Sergio Carbajo AU - Nina Eleni Christou AU - Nicolas Coquelle AU - Eugenio De la Mora AU - Mariam El Khatib AU - Tadeo Moreno Chicano AU - R. Bruce Doak AU - Franck Fieschi AU - Lutz Foucar AU - Oleksandr Glushonkov AU - Alexander Gorel AU - Marie Luise Grünbein AU - Mario Hilpert AU - Mark Hunter AU - Marco Kloos AU - Jason E. Koglin AU - Thomas J. Lane AU - Mengning Liang AU - Angela Mantovanelli AU - Karol Nass AU - Gabriela Nass Kovacs AU - Shigeki Owada AU - Christopher M. Roome AU - Giorgio Schirò AU - Matthew Seaberg AU - Miriam Stricker AU - Michel Thépaut AU - Kensuke Tono AU - Kiyoshi Ueda AU - Lucas M. Uriarte AU - Daehyun You AU - Ninon Zala AU - Tatiana Domratcheva AU - Stefan Jakobs AU - Michel Sliwa AU - Ilme Schlichting AU - Jacques-Philippe Colletier AU - Dominique Bourgeois AU - Martin Weik TI - Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement AID - 10.1101/2021.11.05.462999 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.11.05.462999 4099 - http://biorxiv.org/content/early/2021/11/05/2021.11.05.462999.short 4100 - http://biorxiv.org/content/early/2021/11/05/2021.11.05.462999.full AB - 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 StatementThe authors have declared no competing interest.