Fig. 2: GR interaction with GvTcR and comparison results for mutant isotypes.

a Isothermal titration calorimetry (ITC) analysis shows the binding results for GR with GvTcR. The top and bottom panels show the raw data and the enthalpy changes. The fitting result is shown in the bottom panel as a continuous line. b ITC analysis shows the binding results for R69A/K141A double mutant with GvTcR. These experiments were performed at room temperature. c Protein–protein docking simulations (ClusPro 2.0) were performed to predict the binding of GR and GvTcR. Two amino acid sites capable of polar interaction (R69 and K141) are marked with yellow circles as predictions of binding from the intracellular side. d The absorption spectra of GR and GvTcR were measured by adding GvTcR (ratio 1:1) to observe the spectral shift for 40 min. e The spectral shift of the R69A/K141A double mutant. For the wild type, a blue shift was observed. The mutant GvTcR molar ratio was 1:1, and no shifts were observed. f Light-driven proton pumping activities of GR and GR co-expressed with GvTcR. The pumping data purifies GR from each sample after measurement and calculates the same GR expression ratio. The red-dotted line indicates the pumping activity of GR under white light illumination in the presence of 10 mM NaCl. The black-dotted line represents the GR result upon co-expression with GvTcR. g Time-based kinetic study of purified GR with and without GvTcR showed light-dependent molecular conformational change with a clear accumulation of red-shifted species at approximately 600 nm representing O intermediates, and the depletion of the green region while the dark-adapted sample was used as a baseline for G intermediates. The green line indicates the result for GR + GvTcR, the red line indicates the result for GR + BSA, and the blue line indicates GR. The dotted line indicates the dark condition.