G-protein coupled receptors (GPCRs) regulate a number of physiological processes, where the visual receptor rhodopsin is an excellent prototype to investigate the structural fluctuations underlying GPCR function. Several X-ray crystal structures are available for the dark and active Meta-II states of rhodopsin. However, because of dehydrating conditions used to crystalize the protein, the conformation and orientation of the retinal chromophore in its binding pocket remain uncertain. Our hypothesis was that greater retinal mobility is needed in the active Meta-II state for the interaction with effector proteins. Accordingly, we investigated the structure and orientation of retinal in the dark and the Meta-II states using density functional theory (DFT) calculations and deuterium NMR (<sup>2</sup>H NMR) spectroscopy. For the computational simulations, we modeled the protein environment by including a number of amino acid residues in the retinal binding pocket and the water molecules around it. These models were tested with different chromophore orientations and protonation states of Glu113 and Glu181. Next, we performed DFT and complete active space (CAS) calculations to characterize the electronic structure of the dark and Meta-II states of rhodopsin. <sup>2</sup>H NMR spectroscopic studies were also conducted on the active Meta-II state that contained deuterium-labelled retinal to experimentally validate the possible retinal orientations. The experiment revealed two possible retinal orientations (“flipped” and “unflipped”) without significant steric clashes in the binding pocket of active Meta-II state. By contrast, the DFT calculations show the Meta-II state predominantly contains the “flipped” orientation while the dark-state predominantly accommodates the “unflipped” retinal orientation. Thus the chromophore of the visual receptor undergoes significant mobility changes upon light activation of rhodopsin.

Deuterium NMR spectroscopy and electronic structure calculations capture retinal mobility in rhodopsin activation

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