Visual rhodopsin is an excellent model system to understand the spatio-temporal dynamics of class-A G-protein-coupled receptors (GPCRs). We aim to understand the mechanistic details of how the changes in retinal cofactor propagate to become protein-wide conformational changes over large orders of time. Our hypothesis was that a volumetric expansion occurs in the light-activation process. Here we show the first time-resolved X-ray scattering-based detection of the expansion of rhodopsin leading to the signaling state. We conducted time-resolved small-angle and wide-angle scattering (TR-SAXS and TR-WAXS, respectively) of rhodopsin solubilized in CHAPS detergent at the BioCARS synchrotron beamline (Advanced Photon Source). The photochemistry was initiated with a green (527-nm) laser and probed with X-rays at time delays between 10 ns to 128 ms to collect small and wide-angle difference scattering profiles. Our controls were the retinal-free apoprotein opsin pumped with either green (527-nm) or red (650-nm) light, and the rhodopsin holoprotein pumped with off-resonance red light (650 nm). Optical (pump) laser power titrations were conducted to understand the how visible light modulates the conformational changes following the activation. The absence of difference scattering signals in both controls confirmed the capture of structural information from excitation of the retinal chromophore. Our analysis shows time-dependent increases in the radius of gyration and the volume of the light-activated state versus the inactive dark state which are important for effector proteins to function in visual signaling. The solvent heating found in the high-<i>q </i>region (2–2.5Å<sup>–1</sup>) (TR-WAXS) was used to quantify time-resolved temperature changes in the protein. The analysis showed that multiple photons are absorbed by rhodopsin, which we attribute to sequential absorption by intermediates in the photocycle as the protein traverses its conformational landscape.

Synchrotron x-ray scattering reveals structural dynamics of rhodopsin

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