Rational Control of Off‐State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement**
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- Univ. Grenoble Alpes CEA CNRS Institut de Biologie Structurale F-38044 Grenoble France
- Department of NanoBiophotonics Max Planck Institute for Multidisciplinary Sciences, Göttingen Germany and University Medical Center of Göttingen Clinic for Neurology Göttingen Germany, Fraunhofer Institute for Translational Medicine and Pharmacology ITMP Göttingen Germany
- Max-Planck-Institut für medizinische Forschung Jahnstrasse 29 69120 Heidelberg Germany
- Linac Coherent Light Source (LCLS) SLAC National Accelerator Laboratory 2575, Sand Hill Road Menlo Park CA 94025 USA
- Department of Physics UMR UR1-CNRS 6251 University of Rennes 1 Rennes France
- RIKEN SPring-8 Center Sayo Japan, Japan Synchrotron Radiation Research Institute 1-1-1 Kouto Sayo-cho, Sayo-gun Hyogo 679-5198 Japan
- Institute of Multidisciplinary Research for Advanced Materials Tohoku University Sendai 980-8577 Japan
- Univ. Lille CNRS UMR 8516 LASIR Laboratoire de Spectroscopie pour les Interactions, la Réactivité et l'Environnement Lille 59000 France
- Max-Planck-Institut für medizinische Forschung Jahnstrasse 29 69120 Heidelberg Germany, Department of Chemistry Lomonosov Moscow State University Moscow 119991 Russia
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, 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.
- Research Organization:
- SLAC
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); Max Planck Society; University Grenoble Alpes; French National Research Agency (ANR)
- Grant/Contract Number:
- AC02-76SF00515; P41 GM103393; ANR-17-CE11-0047-01; ANR-17-EURE-0003
- OSTI ID:
- 1881341
- Alternate ID(s):
- OSTI ID: 1886932; OSTI ID: 1891339
- Journal Information:
- ChemPhysChem, Journal Name: ChemPhysChem Vol. 23 Journal Issue: 19; ISSN 1439-4235
- Publisher:
- Wiley Blackwell (John Wiley & Sons)Copyright Statement
- Country of Publication:
- Germany
- Language:
- English
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