1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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The inner mechanics of Rhodopsin Guanylyl Cyclase during cGMP-formation revealed by real-time FTIR spectroscopy

  1. Paul Fischer  Is a corresponding author
  2. Shatanik Mukherjee
  3. Enrico Peter
  4. Matthias Broser
  5. Franz Bartl
  6. Peter Hegemann
  1. Humboldt-Universität zu Berlin, Germany
  2. Center of Advanced European Studies and Research, Germany
  3. Humboldt University of Berlin, Germany
Research Article
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Cite this article as: eLife 2021;10:e71384 doi: 10.7554/eLife.71384

Abstract

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

Data availability

Data files have been provided on Dryad (doi:10.5061/dryad.6wwpzgmzx) for Figure 4 and 5 as well as for the homology structures of the protein presented in the appendices, upon which figure 6 is based. This includes the protein prediction by AlphaFold 2.

The following data sets were generated

Article and author information

Author details

  1. Paul Fischer

    Humboldt-Universität zu Berlin, Berlin, Germany
    For correspondence
    paul.fischer.2@hu-berlin.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3766-9085
  2. Shatanik Mukherjee

    Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7359-9339
  3. Enrico Peter

    Humboldt-Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7913-5597
  4. Matthias Broser

    Humboldt-Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Franz Bartl

    Humboldt-Universität zu Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Peter Hegemann

    Institute of Biology, Experimental Biophysics, Humboldt University of Berlin, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3589-6452

Funding

Deutsche Forschungsgemeinschaft (221545957 B2+B5)

  • Franz Bartl
  • Peter Hegemann

Deutsche Forschungsgemeinschaft (SFB1315-327654276)

  • Peter Hegemann

Deutsche Forschungsgemeinschaft (390540038)

  • Peter Hegemann

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Baron Chanda, Washington University in St. Louis, United States

Publication history

  1. Received: June 17, 2021
  2. Accepted: October 18, 2021
  3. Accepted Manuscript published: October 19, 2021 (version 1)
  4. Accepted Manuscript updated: October 20, 2021 (version 2)
  5. Accepted Manuscript updated: October 25, 2021 (version 3)
  6. Version of Record published: November 8, 2021 (version 4)

Copyright

© 2021, Fischer et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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