The development of X-ray free-electron lasers (XFELs) gives a new avenue to experimentally investigate protein dynamics in femtosecond time regimes. The Rhodopsin family of G-protein-coupled receptors (GPCRs) initiates cellular signaling through nucleotide exchange within G-proteins and are major pharmacological targets worldwide. For GPCRs like visual rhodopsin, an important question is how the local femtosecond dynamics of the ligand initiate the functional protein transitions. However, the extreme reaction speed has been challenging for time-resolved X-ray crystallography. Hence, the structural evidence is sparse in ultrafast timescales. Our research shows 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 pump-probe time-resolved X-ray solution scattering (TR-XSS) studies of detergent-solubilized rhodopsin. Our femtosecond TR-XSS studies using an XFEL detected the protein structural changes without a crystal lattice. We discovered a significant difference-scattering signal within the first few time points immediately following the pump-laser triggering event. Most striking, for rhodopsin in detergent micelles the TR-XSS changes originate in the protein-bound retinal chromophore and are absent for the opsin apoprotein. Two scattering components occur within 10 ps at ambient temperature resulting from retinal isomerization. Furthermore, molecular dynamics (MD) simulations allowed interpretation of the structural motions corresponding to the cis-trans isomerization of retinal within the protein binding pocket. The simulations reveal sub-picosecond motions of conserved amino-acid residues driving transmembrane helical movements. 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. Our experiments directly reveal the initial structural steps of rhodopsin activation and subsequent visual signaling, informing functional dynamics of GPCRs without constraints of crystal packing and cryo-trapping of intermediates.
Menon et al. (Sun,) studied this question.