The development of novel X-ray sources provides a new avenue to experimentally investigate protein dynamics in the picosecond and femtosecond time regimes.  For G-protein-coupled receptors (GPCRs) like rhodopsin, an important question is how the rapid local dynamics of the ligand initiate the larger-scale functional protein transitions. However, structural evidence is lacking 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. Our hypothesis was that rapid <i>cis-trans</i> isomerization of retinal yields ultrafast helical displacements that are propagated by a multiscale mechanism giving large-scale volumetric expansion of rhodopsin in the visual signaling mechanism. To follow the protein structural changes due to light absorption by retinal, we conducted time-resolved pump-probe small-angle X-ray scattering (TR-SAXS) studies of detergent-solubilized rhodopsin.  Femtosecond solution X-ray scattering used an X-ray free-electron laser (XFEL) at LCLS to detect the protein structural change in the absence of a crystal lattice, allowing the first direct detection of <i>cis–trans</i> isomerization of retinal bound to visual rhodopsin in real time. Structural interpretation involved molecular dynamics (MD) simulations which showed that 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. For longer time scales, volumetric changes in the protein were tracked from 10 ns to 128 ms at APS using the pulsed synchrotron X-ray beamline.  Our preliminary analysis reveals time-resolved increases in the radius of gyration and the volume of the light-activated state suggesting it is solvent swollen versus the inactive dark state.  Our time-resolved X-ray scattering experiments directly reveal the initial structural steps of rhodopsin that are important for effector proteins to function in the catalytic reaction mechanism of G-protein activation.

Time-resolved X-ray scattering informs functional dynamics of photoactive membrane protein rhodopsin

ACS Spring 2022

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