Light activation of the visual G-protein-coupled receptor rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane. Enhancement of protein dynamics upon stimulation of the GPCR yields activation of the cognate G-protein (transducin) that initiates biological signaling. Although X-ray crystallographic analysis reveals static structures, changes in protein dynamics are the key to understanding the activation mechanism. Here we show how the integral membrane protein mobility is regulated by the retinal cofactor of the visual GPCR rhodopsin using both elastic and quasi-elastic neutron scattering. Our quasi-elastic neutron scattering (QENS) experiments revealed a logarithmic-like relaxation of the hydrogen-atom dynamics in the Rhodopsin family A GPCRs, as only observed for globular proteins previously. Application of mode-coupling theory (MCT) as originally developed for glass-forming liquids to our QENS analysis reveals the picosecond–nanosecond dynamics in the β-relaxation region crucial to protein function. Our novel powdered GPCR preparation method together with the QENS technique allowed us to uncover subtle changes in protein dynamics regulated by the retinal cofactor of rhodopsin. For the ligand-free opsin apoprotein versus the dark-state rhodopsin, removal of the retinal cofactor increases the relaxation time in the β-relaxation regime (ps–ns), evincing greater protein flexibility. Because opsin is structurally similar to active metarhodopsin-II, which catalytically activates transducin, the cofactor plays a pivotal role in regulating the protein dynamics required for GPCR function.

Visual rhodopsin is an important archetype for G‐protein‐coupled receptors, which are membrane proteins implicated in cellular signal transduction. Herein, we show experimentally that approximately 80 water molecules flood rhodopsin upon light absorption to form a solvent‐swollen active state. An influx of mobile water is necessary for activating the photoreceptor, and this finding is supported by molecular dynamics (MD) simulations. Combined force‐based measurements involving osmotic and hydrostatic pressure indicate the expansion occurs by changes in cavity volumes, together with greater hydration in the active metarhodopsin‐II state. Moreover, we discovered that binding and release of the C‐terminal helix of transducin is coupled to hydration changes as may occur in visual signal amplification. Hydration–dehydration explains signaling by a dynamic allosteric mechanism, in which the soft membrane matter (lipids and water) has a pivotal role in the catalytic G‐protein cycle.

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Knowledge of the activation principles for G-protein-coupled receptors (GPCRs) is critical to development of new pharmaceuticals. Rhodopsin is the archetype for the largest GPCR family, yet the changes in protein dynamics that trigger signaling are not fully understood. Here we show that rhodopsin can be investigated by small-angle neutron scattering (SANS) in fully protiated detergent micelles under contrast matching to resolve lightinduced changes in the protein structure. In SANS studies of membrane proteins, the zwitterionic detergent [(cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) is advantageous because of the low contrast difference between the hydrophobic core and hydrophilic head groups as compared with alkyl glycoside detergents. Combining SANS results with quasielastic neutron scattering reveals how changes in volumetric protein shape are coupled (slaved) to the aqueous solvent. Upon light exposure, rhodopsin is swollen by the penetration of water into the protein core, allowing interactions with effector proteins in the visual signaling mechanism.

Preparation and storage of functional membrane proteins such as G-protein-coupled receptors (GPCRs) are crucial to the processes of drug delivery and discovery. Here we describe a method of preparing powdered GPCRs using rhodopsin as the prototype. We purified rhodopsin in CHAPS detergent with low detergent to protein ratio so the bulk of the sample represented protein (ca. 72% w/w). Our new method for generating powders of membrane proteins followed by rehydration paves the way for conducting functional and biophysical experiments. As an illustrative application powdered rhodopsin was prepared with and without the cofactor 11-cis retinal to enable partial rehydration of the protein with D2O in a controlled manner. Quasielastic neutron scattering studies using both spatial motion and energy landscape models form the basis for crucial insights into structural fluctuations and thermodynamics of GPCR activation.