Cyanobacteriochromes are bistable photoreceptor proteins with desirable photochemical properties for biotechnological applications, such as optogenetics or fluorescence microscopy. Here, we investigate Slr1393-g3, a cyanobacteriochrome that reversibly photoswitches between a red-absorbing (Pr) and green-absorbing (Pg) form. We applied advanced IR spectroscopic methods to track the sequence of intermediates during the photocycle over many orders of magnitude in time. In the conversion from Pg to Pr, we have revealed a new intermediate with distinct spectroscopic features in the IR, which precedes Pr formation using transient IR spectroscopy. In addition, stationary and transient 2D IR experiments measured the vibrational couplings between different groups of the chromophore and the protein in these intermediate states, as well as their structural disorder. Anharmonic QM/MM calculations predict spectra in good agreement with experimental 2D IR spectra of the initial and final states of the photocycle. They facilitate the assignment of the IR spectra that serve as a basis for the interpretation of the spectroscopic results and suggest structural changes of the intermediates along the photocycle.
Bibliographical noteFunding Information:
This work was supported by the Swiss National Science Foundation (SNF) through Grant No. 200020B 188694/1. D.B. acknowledges a Liebig-Scholarship by the funds of the German chemical industry (Fonds der Chemischen Industrie, FCI). I.S. acknowledges support from the Israel Ministry of Science and Technology (Grant 3-16311). I.S. thanks the DFG Collaborative Research Center 1078, project C6, for support. This project emerged during the preparation of the grant application for SNF Sinergia 213507.
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