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A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes

Nature Chemistry volume 7, pages423–430(2015)Cite this article

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Abstract

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto–enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

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Figure 1: Photocycle and structural models of the Pfr state of PaBphP and Agp2.
Figure 2: Experimental and calculated Raman spectra of the Pfr state of Agp2 in the C=C stretching region with different protonation/deuteration patterns of the chromophore.
Figure 3: Infrared difference spectra of Agp2 showing the C=O stretching region for different protonation/deuteration states of the chromophore.
Figure 4: Resonance Raman and infrared difference spectra of Agp2 obtained from samples in H2O.
Figure 5: Effect of pH on the structure and dark reversion kinetics of the Pr state of Agp2.
Figure 6: Keto–enol equilibrium of the Pr chromophore of bathy phytochromes and its role for the Pr to Pfr dark reversion.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft, Sfb1078 (B5, B6, C3). The authors thank the ‘Norddeutscher Verbund für Hoch- und Höchstleistungsrechnen’ (HLRN) for providing computer power.

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Author notes

  1. Francisco Velazquez Escobar and Patrick Piwowarski: These authors contributed equally to this work

Affiliations

  1. Institut für Chemie, Technische Universität Berlin, Sekr. PC 14, Straße des 17. Juni 135, Berlin, D-10623, Germany

    Francisco Velazquez Escobar, Johannes Salewski, Norbert Michael, Maria Fernandez Lopez, Maria Andrea Mroginski & Peter Hildebrandt

  2. Institut für Medizinische Physik und Biophysik, AG Spektroskopie, Charité – Universitätsmedizin Berlin, Charitéplatz 1, Berlin, D-10117, Germany

    Patrick Piwowarski, Anna Rupp & Franz Bartl

  3. Institut für Medizinische Physik und Biophysik, AG Protein X-ray Crystallography and Signal Transduction, Charité – Universitätsmedizin Berlin, Charitéplatz 1, Berlin, D-10117, Germany

    Bilal Muhammad Qureshi & Patrick Scheerer

  4. Abteilung Mikrobiologie, Fachbereich Biologie, Technische Universität Kaiserslautern, Paul-Ehrlich-Straße 23, Kaiserslautern, D-67663, Germany

    Nicole Frankenberg-Dinkel

  5. Institut für Molekulare Medizin und Zellforschung, Sektion Biophysik, Albert-Ludwigs-Universität, Freiburg, Hermann-Herderstraße 9, Freiburg, D-79104, Germany.

    Friedrich Siebert

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  1. Francisco Velazquez Escobar
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Contributions

F.V.E., P.P., M.F.L. and A.R. carried out the RR, infrared and ultraviolet–vis spectroscopic measurements. J.S. and M.A.M. performed and analysed the QM/MM calculations. P.S. provided the homology model for Agp2 and analysed the structural data. B.M.Q. and P.S. provided initial activation assays. F.B. and F.S. analysed the spectroscopic data. P.H. wrote the manuscript with contributions from all authors. The project and experiments were planned and designed by all team members.

Corresponding author

Correspondence to Peter Hildebrandt.

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Velazquez Escobar, F., Piwowarski, P., Salewski, J. et al. A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes. Nature Chem 7, 423–430 (2015). https://doi.org/10.1038/nchem.2225

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