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

Adam, V., Hadjidemetriou, K., Jensen, N., Shoeman, R. L., Woodhouse, J., Aquila, A., et al. (2021). Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement. bioRxiv, 1-24. doi:10.1101/2021.11.05.462999.

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Adam, Virgile, Author
Hadjidemetriou, Kyprianos, Author
Jensen, Nickels, Author
Shoeman, Robert L.1, Author              
Woodhouse, Joyce, Author
Aquila, Andrew, Author
Banneville, Anne-Sophie, Author
Barends, Thomas R. M.1, Author              
Bezchastnov, Victor1, Author              
Boutet, Sébastien, Author
Byrdin, Martin, Author
Cammarata, Marco, Author
Carbajo, Sergio, Author
Christou, Nina Eleni, Author
Coquelle, Nicolas, Author
la Mora, Eugenio De, Author
Khatib, Mariam El, Author
Chicano, Tadeo Moreno, Author
Doak, R. Bruce1, Author              
Fieschi, Franck, Author
Foucar, Lutz1, Author              Glushonkov, Oleksandr, AuthorGorel, Alexander1, Author              Grünbein, Marie Luise1, Author              Hilpert, Mario1, Author              Hunter, Mark, AuthorKloos, Marco1, Author              Koglin, Jason E., AuthorLane, Thomas J., AuthorLiang, Mengning, AuthorMantovanelli, Angela, AuthorNass, Karol1, Author              Nass Kovacs, Gabriela1, Author              Owada, Shigeki, AuthorRoome, Christopher M.1, Author              Schirò, Giorgio, AuthorSeaberg, Matthew, AuthorStricker, Miriam, AuthorThépaut, Michel, AuthorTono, Kensuke, AuthorUeda, Kiyoshi, AuthorUriarte, Lucas M., AuthorYou, Daehyun, AuthorZala, Ninon, AuthorDomratcheva, Tatiana, AuthorJakobs, Stefan, AuthorSliwa, Michel, AuthorSchlichting, Ilme1, Author              Colletier, Jacques-Philippe, AuthorBourgeois, Dominique, AuthorWeik, Martin, Author more..
Affiliations:
1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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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.

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Language(s): eng - English
 Dates: 2021-11-05
 Publication Status: Published online
 Pages: 24
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Title: bioRxiv
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Pages: - Volume / Issue: - Sequence Number: - Start / End Page: 1 - 24 Identifier: -