Recent advances in ultrafast X-ray free-electron lasers (XFELs) have enabled experimental investigations of protein dynamics in the picosecond and femtosecond time regimes. For G-protein-coupled receptors (GPCRs) like rhodopsin—responsible for dim-light vision—an important question is how the rapid local dynamics of the ligand initiate the functional protein transitions. However, structural evidence in the sub-picosecond regime is sparse because until now the extreme reaction speed has precluded direct X-ray diffraction approaches. Here we show how solution X-ray scattering combined with molecular simulations informs the functional dynamics of membrane proteins in ways that could previously only be imagined. We conducted time-resolved pump-probe small-angle X-ray scattering (TR-SAXS) studies of detergent-solubilized rhodopsin to unravel the protein structural changes due to light absorption by retinal. Femtosecond solution X-ray scattering using an XFEL detected rhodopsin structural change in the absence of a crystal lattice, allowing the first direct detection of cis–trans isomerization of retinal bound to visual rhodopsin as it occurred in real-time. We discovered a significant difference-scattering signal following the pump laser triggering event with 100-fs temporal-sampling. Our analysis of the difference scattering profiles showed two dominant states within first 10 picoseconds consistent with photorhodopsin and bathorhodopsin intermediates. Molecular dynamics (MD) simulations furthermore allowed interpretation of the structural motions within the ligand binding pocket corresponding to the cis–trans isomerization of retinal. Upon light excitation, the localized retinal isomerization initiates ultrafast global movements of the transmembrane helices that propagate at the speed of sound throughout the entire protein. The overall volumetric expansion is driven by the global structural changes over longer time scales that trigger the visual signaling cascade. Our experiments directly reveal the global and local motions of rhodopsin that are precursors to functional G-protein activation, and subsequent visual signaling.

Suchithranga M.D.C. Perera

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