Abstract
The function of photoreceptors relies on efficient transfer of absorbed light energy from the chromophore to the protein to drive conformational changes that ultimately generate an output signal. In retinal-binding proteins, mainly two mechanisms exist to store the photon energy after photoisomerization: 1) conformational distortion of the prosthetic group retinal, and 2) charge separation between the protonated retinal Schiff base (RSBH+) and its counterion complex. Accordingly, energy transfer to the protein is achieved by chromophore relaxation and/or reduction of the charge separation in the RSBH+-counterion complex. Combining FTIR and UV-Vis spectroscopy along with molecular dynamics simulations, we show here for the widely used, red-activatable Volvox carteri channelrhodopsin-1 derivate ReaChR that energy storage and transfer into the protein depends on the protonation state of glutamic acid E163 (Ci1), one of the counterions of the RSBH+. Ci1 retains a pKa of 7.6 so that both its protonated and deprotonated forms equilibrate at physiological conditions. Protonation of Ci1 leads to a rigid hydrogen-bonding network in the active-site region. This stabilizes the distorted conformation of the retinal after photoactivation and decelerates energy transfer into the protein by impairing the release of the strain energy. In contrast, with deprotonated Ci1 or removal of the Ci1 glutamate side chain, the hydrogen-bonded system is less rigid, and energy transfer by chromophore relaxation is accelerated. Based on the hydrogen out-of-plane (HOOP) band decay kinetics, we determined the activation energy for these processes in dependence of the Ci1 protonation state.
| Original language | American English |
|---|---|
| Pages (from-to) | 705-716 |
| Number of pages | 12 |
| Journal | Biophysical Journal |
| Volume | 119 |
| Issue number | 3 |
| DOIs | |
| State | Published - 4 Aug 2020 |
Bibliographical note
Funding Information:This work was funded by the Deutsche Forschungsgemeinschaft via Sonderforschungsbereich 1078, project B2 (P.H.) and B5 (F.J.B.) and the Cluster of Excellence 314 “Unifying Concepts in Catalysis” (project E4/D4 to P.H.). E.R. acknowledges support by the Bundesministerium für Bildung und Forschung grant 05K16KH1 . P.H. is a Hertie Senior Professor for Neuroscience and was supported by the Hertie-Foundation . S.A. acknowledges support by the Minerva Stiftung for a postdoctoral fellowship. I.S. thanks the SFB 1078 “Protonation Dynamics in Protein Function” for support by a Mercator fellowship. I.S. gratefully acknowledges funding by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant no. 678169 “PhotoMutant”).
Funding Information:
We thank Christina Schnick, Anja Koch, and Katja Stehfest for protein expression in P. pastoris, Paul Fischer for contributing to the evaluation software, and Thomas P. Sakmar for providing the 1D4 antibody. This work was funded by the Deutsche Forschungsgemeinschaft via Sonderforschungsbereich 1078, project B2 (P.H.) and B5 (F.J.B.) and the Cluster of Excellence 314 ?Unifying Concepts in Catalysis? (project E4/D4 to P.H.). E.R. acknowledges support by the Bundesministerium f?r Bildung und Forschung grant 05K16KH1. P.H. is a Hertie Senior Professor for Neuroscience and was supported by the Hertie-Foundation. S.A. acknowledges support by the Minerva Stiftung for a postdoctoral fellowship. I.S. thanks the SFB 1078 ?Protonation Dynamics in Protein Function? for support by a Mercator fellowship. I.S. gratefully acknowledges funding by the European Research Council under the European Union's Horizon 2020 research and innovation program (grant no. 678169 ?PhotoMutant?).
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