Small-Angle Neutron Scattering Reveals Energy Landscape for Rhodopsin Photoactivation

Abstract

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.

Differentiating between Inactive and Active States of Rhodopsin by Atomic Force Microscopy in Native Membranes

“…Light-activation of rhodopsin is known to allow water to access the interior of the rhodopsin molecule, allowing the hydrolysis of the chromophore 38 . A study using small-angle neutron scattering with quasielastic neutron scattering indicated that light-activation of rhodopsin results in the penetration of water into the interior of the receptor, which results in a more tightly packed interior and more compact core structure that can facilitate the movement of transmembrane helices and coupling to signaling partners 39 . This compact core would exhibit higher stiffness when pressed by the AFM probe.…”

Activation of the G‐Protein‐Coupled Receptor Rhodopsin by Water

“…By conducting experiments for rhodopsin solubilized in micelles of detergents such as [(cholamidopropyl)dimethylammonio]‐propanesulfonate (CHAPS) or n ‐dodecyl‐β‐ d ‐maltoside (DDM), one can isolate the protein hydration effect from the lipid contribution. Direct comparability with recent small‐angle neutron scattering (SANS) [16c] and quasi‐elastic neutron scattering (QENS) studies of rhodopsin hydration is then possible, which support water uptake in the active MII state. In this regard, experimental SDSL studies [29a] of rhodopsin in DDM micelles have shown that the small osmolyte sucrose back shifts the population toward the MI component.…”

“…Our findings reveal how the soft cellular matter comprising the water and lipids is likely to affect the transmembrane signaling in the case of rhodopsin. Integrating the current results with small‐angle neutron scattering (SANS) studies of rhodopsin in detergent solutions, [16c] and quasi‐elastic neutron scattering (QENS) data for partially hydrated powders, [43] affords new insights into an energy landscape mechanism versus a bimodal‐switch model. According to our picture, rhodopsin is swollen by penetration of water into the protein core following the light exposure [12a, 16c, 43] .…”

“…Integrating the current results with small‐angle neutron scattering (SANS) studies of rhodopsin in detergent solutions, [16c] and quasi‐elastic neutron scattering (QENS) data for partially hydrated powders, [43] affords new insights into an energy landscape mechanism versus a bimodal‐switch model. According to our picture, rhodopsin is swollen by penetration of water into the protein core following the light exposure [12a, 16c, 43] . Volumetric changes in protein shape are coupled (slaved) to the aqueous solvent, including the hydration shell and bulk solvent [16c, 44] .…”

Neutron scattering in the biological sciences: progress and prospects

“…SANS requires quantities of purified proteins comparable to those for crystallographic trials (1 mg per sample). Membrane-protein biochemists, biophysicists and structural biologists can readily take advantage of SANS and contrast variation to obtain coveted three-dimensional structural models of detergent-, vesicle-, bicelle-or nanodiscstabilized membrane proteins (Bayburt et al, 2002;Nath et al, 2007;Ritchie et al, 2009;Perera et al, 2018) in solution and in complex with binding partners or substrates (Breyton et al, 2013;Trewhella, 2006). The software required (Pé rez & Koutsioubas, 2015) differs only slightly from the SAXS software that is familiar to structural biologists.…”