BPS2026 – Deciphering retinal orientation and dynamics in rhodopsin with deuterium NMR and quantum mechanical calculations

G-protein-coupled receptors (GPCRs) mediate various physiological processes, among which the archetypical visual receptor protein, rhodopsin, yields deep insight into understanding the structural mechanisms of class A GPCRs. Although X-ray crystal structures of rhodopsin’s photoactivated metarhodopsin-II (Meta-II) state exist, the exact structural and orientational dynamics of the retinal chromophore remain unclear. 1 Here, we employed deuterium nuclear magnetic resonance ( 2 H NMR) spectroscopy and electronic structure calculations to pinpoint the orientation and mobility within the active Meta-II state. Using selectively deuterium-labeled methyl groups along retinal’s polyene chain (at C5, C9, or C13), we acquired experimental 2 H NMR spectra. Identifying the bond orientation of the labeled groups revealed the chromophore’s alignment within the binding site. We discovered two plausible retinal orientations within the Meta-II binding pocket, “flipped” and “unflipped,” both avoiding steric hindrance from surrounding residues. Computational models assumed polar interactions from ten local amino-acid residues, deemed quantitatively sufficient to balance accuracy with computational efficiency. Water molecules in varying proportions were also distributed throughout the active environment providing additional polar contacts within the binding site. Lastly, we simulated events in Schiff base deprotonation with respect to candidate counterions (i.e., E113 and E181). Preliminary time-dependent density functional theory (TD-DFT) calculations identified key molecular orbitals involved in the electronic transition, followed by complete active-space (CAS) calculations to determine precise absorption spectra. The procedure was repeated independently for monomers and homodimers for completeness and accuracy. Notably, the results suggest the relative favorability of the flipped orientation upon activated retinal’s transition from the preactive Meta-I intermediate to active Meta-II. Together, our spectroscopic and computational approaches illuminate the light-induced structural dynamics of retinal, offering broad insight into the mechanisms underlying GPCR activation. 1 Ryazantsev, M.N. et al., (2019). J. Membr. Biol. 252, 425–449.