Powdered G-Protein-Coupled Receptors

Abstract

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.

Small-Angle Neutron Scattering Reveals Energy Landscape for Rhodopsin Photoactivation

“…Our hypothesis was that light absorption gives a flood of bulk water into rhodopsin, underlying the catalytic binding and unbinding of the G-protein transducin. Through an integrative approach, combining small-angle neutron scattering (SANS) with quasielastic neutron scattering (QENS), , we discovered how the protein dynamics are coupled (slaved) to the hydration shell and to the bulk solvent. , Interactions between the rhodopsin molecules in detergent micelles upon light activation characterize the energy landscape (EL) for protein function. Greater hydrated volume accompanies water penetration into the rhodopsin core, yielding a solvent-swollen state that facilitates coupling with effector proteins such as transducin or arrestin.…”

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

“…Upon photon absorption, the elimination of voids or cavities due to the water penetration yields a solvent‐swollen protein interior, with a further increase of hydration in the active MII state [12] . Neutron scattering studies, [16c, 43b] as well as hydrostatic pressure [45] and osmotic pressure measurements, thus point to greater hydrated volume upon forming the active MII state in the reaction mechanism [5b]…”

“…Indeed, the force‐based measurements give evidence of a functional volumetric change that occurs in conjunction with light activation of rhodopsin. We propose that formation of active MII entails an interplay of hydration and packing forces, [16c, 43b] whereby the penetration of water into the protein core yields a solvent‐swollen active state. Changes of helix packing density affect small voids or cavities within the protein, as described by Richards [48] and by Hummer et al [49] .…”

“…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 ab imodal-switch model. According to our picture, rhodopsin is swollen by penetration of water into the protein core following the light exposure.…”

Dynamics of Apolipoprotein B-100 in Interaction with Detergent Probed by Incoherent Neutron Scattering

“…Moreover, deuterated detergents remain an expensive solution, and the proposed protocols are not applicable to all membrane proteins. Recently, a QENS study on the G-protein-coupled receptor was conducted by Shrestha et al, , whose sample contained 25% of the remaining detergent. In that specific case, decoupling dynamics between protein and detergent made the detergent correction straightforward and the extraction of the protein contribution successful.…”