Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Abstract Determining the molecular origin of spectral tuning in photoactive biological systems is instrumental for understanding their function. Spectral-tuning efforts for flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, are limited by their dependency on protein-bound flavins, whose structure and hence electronic properties, cannot be altered by mutation. To address those shortcomings, we here present the photophysical, computational and structural characterization of structurally uncharacterized blue-shifted FbFPs, carrying a previously described lysine substitution within their flavin-binding pocket. X-ray structures reveal displacement of the lysine away from the chromophore and opening up of the structure as cause for the blue shift. Site-saturation mutagenesis and high-throughput screening, yielded a red-shifted variant, in which the lysine side chain of the blue-shifted variant is stabilized in close distance to the flavin by a secondary mutation, mechanistically accounting for the red shift. Thus, a single secondary mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools that should find application in life sciences.
Authors 16
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Forschungszentrum Jülich · Heinrich Heine University Düsseldorf
Affiliation as printed
Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
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Affiliation as printed
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
JuStruct: Jülich Center for Structural Biology, Forschungszentrum Jülich, 52428 Jülich, Germany
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-545 Jülich, Germany
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Moscow Institute of Physics and Technology
Affiliation as printed
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 11701 Dolgoprudny, Russia
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Affiliation as printed
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074 Aachen, Germany
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-2074 Aachen, Germany
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Affiliation as printed
IBI-1: Molecular and Cellular Physiology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
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Moscow Institute of Physics and Technology
Affiliation as printed
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 11701 Dolgoprudny, Russia
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Centre National de la Recherche Scientifique · Moscow Institute of Physics and Technology · Forschungszentrum Jülich · Commissariat à l'Énergie Atomique et aux Énergies Alternatives · Institut de Biologie Structurale · RWTH Aachen University · Université Grenoble Alpes
Affiliation as printed
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
Institut de Biologie Structurale Jean-Pierre Ebel, Université Grenoble Alpes-Commissariat à l’Energie Atomique et aux Energies Alternatives–CNRS, 38044 Grenoble, France
Institute of Crystallography, RWTH Aachen University, 52062 Aachen, Germany
JuStruct: Jülich Center for Structural Biology, Forschungszentrum Jülich, 52428 Jülich, Germany
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-545 Jülich, Germany
Institut de Biologie Structurale Jean-Pierre Ebel, Université Grenoble Alpes-Commissariat à l'Energie Atomique et aux Energies Alternatives–CNRS, 3044 Grenoble, France
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 11701 Dolgoprudny, Russia
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Moscow Institute of Physics and Technology
Affiliation as printed
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 11701 Dolgoprudny, Russia
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Moscow Institute of Physics and Technology
Affiliation as printed
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 11701 Dolgoprudny, Russia
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Forschungszentrum Jülich · Heinrich Heine University Düsseldorf
Affiliation as printed
IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
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Ulrich Schwaneberg Aachen DWI - Leibniz Institute for Interactive Materials Institute of Biotechnology
DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University
Affiliation as printed
DWI-Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, 52056, Aachen, Germany
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074 Aachen, Germany
Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-2074 Aachen, Germany
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Centre National de la Recherche Scientifique · Moscow Institute of Physics and Technology · Forschungszentrum Jülich · Commissariat à l'Énergie Atomique et aux Énergies Alternatives · Institut de Biologie Structurale · Université Grenoble Alpes
Affiliation as printed
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
Institut de Biologie Structurale Jean-Pierre Ebel, Université Grenoble Alpes-Commissariat à l’Energie Atomique et aux Energies Alternatives–CNRS, 38044 Grenoble, France
JuStruct: Jülich Center for Structural Biology, Forschungszentrum Jülich, 52428 Jülich, Germany
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-545 Jülich, Germany
Institut de Biologie Structurale Jean-Pierre Ebel, Université Grenoble Alpes-Commissariat à l'Energie Atomique et aux Energies Alternatives–CNRS, 3044 Grenoble, France
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 11701 Dolgoprudny, Russia
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Forschungszentrum Jülich · Heinrich Heine University Düsseldorf
Affiliation as printed
IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
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Affiliation as printed
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
JuStruct: Jülich Center for Structural Biology, Forschungszentrum Jülich, 52428 Jülich, Germany
IBI-7: Structural Biochemistry, Forschungszentrum Jülich GmbH, D-545 Jülich, Germany
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Ivan Gushchin corresponding
Moscow Institute of Physics and Technology
Affiliation as printed
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Russia
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, 11701 Dolgoprudny, Russia
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Ulrich Krauß corresponding
Forschungszentrum Jülich · Heinrich Heine University Düsseldorf
Affiliation as printed
IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
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