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Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species

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Abstract

Light-oxygen-voltage (LOV) domains are common photosensory modules that found many applications in fluorescence microscopy and optogenetics. Here, we show that the Chloroflexus aggregans LOV domain can bind different flavin species (lumichrome, LC; riboflavin, RF; flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) during heterologous expression and that its physicochemical properties depend strongly on the nature of the bound flavin. We show that whereas the dissociation constants for different chromophores are similar, the melting temperature of the protein reconstituted with single flavin species varies from ~ 60 °C for LC to ~ 81 °C for FMN, and photobleaching half-times vary almost 100-fold. These observations serve as a caution for future studies of LOV domains in non-native conditions yet raise the possibility of fine-tuning various properties of LOV-based fluorescent probes and optogenetic tools by manipulating the chromophore composition.

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Abbreviations

CFP:

Cyan fluorescent protein

FAD:

Flavin adenine dinucleotide

FbFP:

Flavin-based fluorescent protein

FMN:

Flavin mononucleotide

GFP:

Green fluorescent protein

HPLC:

High-performance liquid chromatography

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

LC:

Lumichrome

LOV:

Light-oxygen-voltage

PAS:

Per-Arnt-Sim

RF:

Riboflavin

RFP:

Red fluorescent protein

SEC:

Size-exclusion chromatography

YFP:

Yellow fluorescent protein

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Acknowledgements

We are grateful to Mikhail Shevtsov and Tatiana Kotova (Moscow Institute of Physics and Technology) for help with HPLC experiments and Nina Malyar (Moscow Institute of Physics and Technology) for help with pH titration experiments. We are grateful to Thomas Drepper (Institute of Molecular Enzyme Technology, HHU Düsseldorf, FZ-Jülich) for sharing the data on thermal stability of FbFPs from thermophilic microorganisms and Esther Knieps-Grünhagen (Institute of Molecular Enzyme Technology, HHU Düsseldorf, FZ-Jülich) for help with protein purification. The work was supported by the Russian Science Foundation, grant number 21-64-00018.

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Authors and Affiliations

  1. Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia

    Anastasia Smolentseva, Ivan M. Goncharov, Anna Yudenko, Andrey Bogorodskiy, Oleg Semenov, Vera V. Nazarenko, Valentin Borshchevskiy, Alina Remeeva, Valentin Gordeliy & Ivan Gushchin

  2. Institute of Cytology, Russian Academy of Sciences, 194064, Saint Petersburg, Russia

    Alexander V. Fonin

  3. Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, Forschungszentrum Jülich, Jülich, Germany

    Karl-Erich Jaeger & Ulrich Krauss

  4. Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany

    Karl-Erich Jaeger & Ulrich Krauss

  5. Institut de Biologie Structurale J.-P. Ebel, Université Grenoble Alpes-CEA-CNRS, 38000, Grenoble, France

    Valentin Gordeliy

  6. Institute of Biological Information Processing IBI-7: Structural Biochemistry, Forschungszentrum Jülich, 52428, Jülich, Germany

    Valentin Borshchevskiy & Valentin Gordeliy

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  1. Anastasia Smolentseva
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  2. Ivan M. Goncharov
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  3. Anna Yudenko
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Correspondence to Ivan Gushchin.

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Smolentseva, A., Goncharov, I.M., Yudenko, A. et al. Extreme dependence of Chloroflexus aggregans LOV domain thermo- and photostability on the bound flavin species. Photochem Photobiol Sci 20, 1645–1656 (2021). https://doi.org/10.1007/s43630-021-00138-3

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