Hydration Thermodynamics of a Powdered G-Protein-Coupled Receptor

Abstract

Membrane proteins such as G protein-coupled receptors (GPCRs) are prominent targets for novel pharmaceutical drugs. Preparation and storage of fully functional GPCRs is crucial to the processes of drug delivery and discovery. Here we describe a novel method of preparing powdered GPCRs using rhodopsin as the prototype. Our new method for generating powdered samples, notable for exceptionally high protein content while retaining photochemical functionality, paves the way for conducting functional and biophysical experiments on the dynamic process of receptor activation. As an illustrative application, powdered rhodopsin is prepared with and without the cofactor 11-cis retinal enabling the partial hydration of the protein with 2H2O in a controlled manner [1]. These samples were used to study the changes in hydrogen-atom dynamics using the quasi-elastic neutron scattering (QENS) technique. Evaluation of QENS data using the interpretations of both spatial motion and energy landscape models offer key insights into the thermodynamics of GPCR activation. The QENS studies reveal a broadly distributed relaxation of the hydrogen atom dynamics of rhodopsin on a picosecond-nanosecond time scale, essential for protein function, previously observed for only globular proteins [2]. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differences can be attributed to the influence of the covalently bound retinal ligand. Furthermore, preparation of powdered samples from photoilluminated rhodopsin effectively captures the state of the excited protein while undergoing the conformational changes leading to activation. These discoveries create new avenues towards understanding the central role of water in GPCR activation and unlocking the mechanisms of the biologically important signaling process of rhodopsin. [1] S.M.D.C. Perera et al. (2016) J. Phys. Chem. Lett.7, 4239-4235. [2] U.R. Shresta et al. (2016) J. Phys. Chem. Lett. 7, 4130-4136.