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Chromophore reduction plus reversible photobleaching: how the mKate2 “photoconversion” works

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Abstract

mKate red-to-green photoconversion is a non-canonical type of phototransformation in fluorescent proteins, with a poorly understood mechanism. We have hypothesized that the daughter mKate2 protein may also be photoconvertible, and that this phenomenon would be connected with mKate(2) chromophore photoreduction. Indeed, upon the intense irradiation of the protein sample supplemented by sodium dithionite, the accumulation of green as well as blue spectral forms is enhanced. The reaction was shown to be reversible upon the reductant’s removal. However, an analysis of the fluorescence microscopy data, absorption spectra, kinetics and time-resolved fluorescence spectroscopy revealed that the short-wavelength spectral forms of mKate(2) exist before photoactivation, that their fractions increase light-independently after dithionite addition, and that the conversion is facilitated by the photobleaching of the red chromophore form.

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Supplementary information is available along with an online-version of the manuscript at https://doi.org/10.1007/s43630-021-00060-8. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

This study was supported by the Russian Science Foundation (RSCF), Grant № 20-14-00255. The funders of the study had no part in the writing, analysis, interpretation, or decision to submit the paper. Experiments were partially carried out using equipment provided by the IBCH core facility (CKP IBCH, supported by Russian Ministry of Education and Science, Grant RFMEFI62117X0018).

Funding

This study was supported by the Russian Science Foundation (RSCF), Grant № 20–14-00255.

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Authors and Affiliations

  1. Faculty of Biology, Lomonosov Moscow State University, 119992, Moscow, Russia

    Elena A. Protasova & Eugene G. Maksimov

  2. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997, Moscow, Russia

    Alexander S. Mishin, Konstantin A. Lukyanov & Alexey M. Bogdanov

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  1. Elena A. Protasova
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  2. Alexander S. Mishin
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  3. Konstantin A. Lukyanov
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  4. Eugene G. Maksimov
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EAP: investigation, formal analysis, software; ASM: validation, investigation; KAL: conceptualization, supervision, resources; EGM: methodology, validation, formal analysis, investigation, visualization; AMB: conceptualization, investigation, project administration, funding acquisition. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Correspondence to Alexey M. Bogdanov.

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Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer - a topical issue in honour of Klaus Brettel.

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Protasova, E.A., Mishin, A.S., Lukyanov, K.A. et al. Chromophore reduction plus reversible photobleaching: how the mKate2 “photoconversion” works. Photochem Photobiol Sci 20, 791–803 (2021). https://doi.org/10.1007/s43630-021-00060-8

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