Investigation of Rhodopsin Activation Using Spectroscopic and Scattering Techniques

Abstract

G-protein–coupled receptors are the largest superfamily in the human genome, and involved in critical cellular signaling processes in living cells. Protein structural fluctuations are the key for GPCR function that is driven and modulated by a variety of factors that are not well understood. This dissertation focusses on understanding the activation of GPCRs using the visual receptor, rhodopsin as the prototype. Rhodopsin is an ideal candidate for this study, as it represents the largest class of GPCRs, and is known to demonstrate more noticeable structural changes upon activation compared to the other GPCRs. What structural fluctuations occur, the role of water, and how the retinal cofactor regulates the protein dynamics during rhodopsin activation are specific research problems addressed in this work. Hypothesizing an ensemble activation mechanism, experiments were conducted using a variety of techniques to probe structural and dynamical fluctuations of rhodopsin in native membranes, as well as in membrane mimetics such as detergent micelles. Time-resolved wide-angle X-ray scattering (TR-WAXS), small-angle neutron scattering (SANS), quasielastic neutron scattering (QENS), and electronic spectroscopy are among the prominent techniques used to gain insights into the photo-intermediates that are key to understanding the rhodopsin activation process. The small-angle neutron scattering (SANS) experiments revealed a volumetric expansion of the protein molecule upon photoactivation of rhodopsin. Electronic spectroscopy together with the differential hydration study revealed the crucial role of water in rhodopsin signaling process and signal amplification by water. The quasielastic neutron scattering study conducted on powdered rhodopsin probed the changes in the local dynamics that are regulated by the retinal cofactor of the rhodopsin molecule. The increased local steric crowding in the ligand-free opsin is consistent with collapsing of the apoprotein structure in the absence of the retinal chromophore leading to inactive opsin conformation. Finally, a time-resolved wide-angle X-ray scattering study was conducted using the X-ray free electron laser at the SLAC national laboratory to probe the early structural fluctuations in rhodopsin photoactivation. The preliminary pump-probe experiments conducted on rhodopsin in CHAPS detergent micelles revealed a light-triggered protein quake that occurs during the early activation stages of rhodopsin photoactivation. Thus the protein fluctuations underlying the GPCR function are revealed by neutrons, X-rays, and other photons in a combined implementation of both spectroscopic and scattering techniques as applied to the investigation of rhodopsin activation.