Abstract
G-protein-coupled receptors of the Rhodopsin family are implicated in transmitting signals across biomembranes and are among the most widespread pharmaceutical targets. For G-protein-coupled receptors (GPCRs) like rhodopsin, an important question is how the rapid local dynamics of the ligand initiate the functional protein transitions. Despite the availability of crystal structures of rhodopsin and cryo-trapped intermediates, it is unclear how the protein dynamics are affected by lattice-packing forces or low temperatures. We hypothesize that the crystalline environment inhibits the full range of conformational motions in the visual activation. Here we show how solution X-ray scattering combined with molecular simulations informs the functional dynamics of membrane proteins. To follow the protein structural changes due to light absorption by retinal, we conducted time-resolved X-ray solution scattering (TR-XSS) studies of detergent-solubilized rhodopsin. Femtosecond solution X-ray scattering using an X-ray free electron laser (XFEL) detected the protein structural change in the absence of a crystal lattice and allowed the first direct observation of cis-trans isomerization of retinal bound to visual rhodopsin as it occurred in real time [1]. We discovered a significant difference-scattering signal within the first few time points immediately following the pump laser triggering event. Two scattering components were found within 10 ps after photoactivation at ambient temperature. The first was consistent with structurally similar photo/bathorhodopsin intermediates, and the second due to changes in solvent dynamics. Interpretation of the TR-XSS profiles using molecular dynamics simulations reveal significant atomic displacements in the β-ionone ring, and the amino acids like Tyr268 previously thought to occur later in the photocycle. Our findings demonstrate how X-ray scattering with an XFEL informs the functional dynamics of GPCRs, without artifacts caused by crystal packing or cryo-trapping photointermediates. [1] M.F. Brown et al. (2022) Biophys. J. 121, 193a.
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