Neutron Scattering Reveals Protein Fluctuations in GPCR Activation

Abstract

Protein fluctuations are the key for activation of G-protein-coupled receptors (GPCRs) such as rhodopsin. X-ray crystallography reveals useful structural information about the intermediates in the photoactivation process; however, knowledge of protein dynamical changes is pivotal for understanding the activation mechanism of GPCRs. We hypothesized that rhodopsin activation leads to multiple activated states in accord with an ensemble-activation mechanism (EAM) [1]. Neutron scattering provides us with non-invasive techniques to study both structural and dynamical transitions associated with the function of physiologically important proteins such as rhodopsin. Here we describe a combined small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS) approach to investigate the structural fluctuations associated with rhodopsin activation. The samples were prepared by purifying rhodopsin in detergents such as CHAPS from rhodopsin disk membranes. In SANS the intensity of the neutrons scattered is measured as a function of momentum transfer (Q). The experiments conducted on detergent contrast-matched conditions allowed us to probe the light-induced structural changes in rhodopsin exclusively in protein-detergent micelles. The SANS study unveiled a volumetric expansion of the protein upon photoactivation of rhodopsin. In QENS, the energy spectrum of the neutron scattered as a function of transfer-energy (ω) is measured for partially hydrated (h ≈ 0.27) rhodopsin samples. The QENS data in conjunction with the mode-coupling theory (MCT) revealed that the β-fluctuations in ligand-free opsin are substantially slower than in the dark-state rhodopsin, which indicates an increase in protein flexibility upon rhodopsin photoactivation. The volumetric expansion (from SANS experiments) and increased protein flexibility (from QENS experiments) are consistent with an increase in the number of configurations upon rhodopsin photoactivation as previously suggested by an EAM. Hence, neutron scattering provides insights into protein fluctuations crucial for GPCR activation. [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268.