G-Protein-Coupled Receptor Activation Investigated using Small-Angle Neutron Scattering

Abstract

G-protein-coupled receptors (GPCRs) represent the largest family of proteins in the human genome and comprise about 50% of current molecular drug targets. Rhodopsin is the GPCR involved in visual light perception and occurs naturally in a membrane lipid environment. Rhodopsin photoactivation yields cis-trans isomerization of retinal giving an equilibrium between inactive Meta-I and active Meta-II states. Here we address the question: does photoactivation lead to a single Meta-II conformation or is there an ensemble of substates described by an ensemble-activation mechanism (EAM) [1]? In this context small-angle neutron scattering (SANS) probes rhodopsin-detergent and rhodopsin-lipid complexes through measurement of the intensity of the neutrons scattered as a function of scattering vector I(q). Contrast variation enables us to highlight individual components of a multi-component system without isotopic labeling of the sample. Upon photoactivation, the Meta-I state was stabilized in CHAPS-solubilized rhodopsin, while Meta-II was trapped in DDM-solubilized rhodopsin. The ligand-free apoprotein opsin was obtained by photobleaching rhodopsin in the presence of hydroxylamine. The SANS spectra for the above rhodopsin substates were acquired from 80% D2O solutions and at contrast-matching points for both DDM and CHAPS samples. The data collected in 80% D2O samples provide structural information for both protein and detergent, while the data collected at contrast-matching points give information for the protein structure exclusively. Our experiments demonstrate that for detergent-solubilized rhodopsin, SANS with contrast variation can detect structural differences between the rhodopsin dark-state, Meta-I, Meta-II, and ligand-free opsin states. Dark-state rhodopsin is more conformationally flexible (less-compact) in DDM micelles compared to the CHAPS, which is consistent with an ensemble of activated Meta-II states. Furthermore, the time-dependent structural transitions between Meta-I and Meta-II as observed by time-resolved SANS will be crucial to understanding the ensemble-based activation. [1]A.V. Struts et al. (2011) NSMB18, 392-394.