Quasi-elastic Neutron Scattering Reveals Ligand-Induced Protein Dynamics of a G-Protein-Coupled Receptor

Abstract

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.

Powdered G-Protein-Coupled Receptors

“…26 Here, we show the use of the powdered GPCR preparation method to investigate rhodopsin versus the ligand-free opsin apoprotein. Rhodopsin is a class A GPCR responsible for vision under dim light conditions in vertebrates.…”

“…Current findings suggest the intrinsic dynamics of the protein are unlocked by the light-induced isomerization of the 11- cis retinal cofactor needed for interaction of the GPCR with its cognate G-protein (transducin). 26 …”

“…44 Quasielastic neutron scattering methods enable one to probe molecular motions from picoseconds up to nanoseconds within the atomic to molecular length scales. 26 Upon hydrating the protein sample with D 2 O, the results of a QENS experiment are expressed as the dynamic structure factor, which is dominated by the contributions from non-exchangeable hydrogen atoms in the protein. The QENS data can be analyzed both in the energy domain by a model-free analysis, and in the time domain by introduction of motional models.…”

“…26 For D 2 O-hydrated powders of rhodopsin/CHAPS detergent, the majority of the recorded signal is due to the protein dynamics, and the correction for the presence of the detergent is minimal during the subsequent data reduction and analysis. The QENS experiments were focused on studying how the dynamics of the protein were affected upon rhodopsin photoactivation.…”

“…The QENS experiments were focused on studying how the dynamics of the protein were affected upon rhodopsin photoactivation. Protein dynamics of the dark-state rhodopsin were compared to those of ligand-free opsin, 26 which is structurally similar to active metarhodopin-II. 38 Previous neutron scattering studies conducted to unravel the local dynamics of bacteriorhodopsin and visual rhodopsin in the dark and light-activated states have revealed few significant differences.…”