학술논문
Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.
Document Type
Academic Journal
Author
Adam V; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Hadjidemetriou K; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Jensen N; 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.; Shoeman RL; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Woodhouse J; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Aquila A; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Banneville AS; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Barends TRM; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Bezchastnov V; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Boutet S; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Byrdin M; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Cammarata M; Department of Physics, UMR UR1-CNRS 6251, University of Rennes 1, Rennes, France.; Carbajo S; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Eleni Christou N; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Coquelle N; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; De la Mora E; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; El Khatib M; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Moreno Chicano T; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Bruce Doak R; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Fieschi F; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Foucar L; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Glushonkov O; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Gorel A; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Grünbein ML; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Hilpert M; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Hunter M; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Kloos M; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Koglin JE; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Lane TJ; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Liang M; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Mantovanelli A; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Nass K; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Nass Kovacs G; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Owada S; RIKEN SPring-8 Center, Sayo, Japan.; Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo, 679-5198, Japan.; Roome CM; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Schirò G; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Seaberg M; Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, 2575, Sand Hill Road, Menlo Park, CA, 94025, USA.; Stricker M; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Thépaut M; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Tono K; RIKEN SPring-8 Center, Sayo, Japan.; Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo, 679-5198, Japan.; Ueda K; Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, 980-8577, Japan.; Uriarte LM; Univ. Lille, CNRS, UMR 8516, LASIR, Laboratoire de Spectroscopie pour les Interactions, la Réactivité et l'Environnement, Lille, 59000, France.; You D; Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, 980-8577, Japan.; Zala N; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Domratcheva T; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Department of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia.; Jakobs S; 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.; Sliwa M; Univ. Lille, CNRS, UMR 8516, LASIR, Laboratoire de Spectroscopie pour les Interactions, la Réactivité et l'Environnement, Lille, 59000, France.; Schlichting I; Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, 69120, Heidelberg, Germany.; Colletier JP; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Bourgeois D; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.; Weik M; Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale, F-38044, Grenoble, France.
Source
Publisher: Wiley-VCH Verlag Country of Publication: Germany NLM ID: 100954211 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1439-7641 (Electronic) Linking ISSN: 14394235 NLM ISO Abbreviation: Chemphyschem Subsets: MEDLINE
Subject
Language
English
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.
(© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)
(© 2022 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)