Protein Sequence and Membrane Lipid Roles in the Activation Kinetics of Bovine and Human Rhodopsins

Rhodopsin is a G protein-coupled receptor found in the rod outer segments in the retina, which triggers a visual response under dim light conditions. Recently, a study of the late, microsecond-to-millisecond kinetics of photointermediates of the human and bovine rhodopsins in their native membranes revealed a complex, double-square mechanism of rhodopsin activation. In this kinetic scheme, the human rhodopsin exhibited more Schiff base deprotonation than bovine rhodopsin, which could arise from the ∼7% sequence difference between the two proteins, or from the difference between their membrane lipid environments. To differentiate between the effects of membrane and protein structure on the kinetics, the human and bovine rhodopsins were inserted into 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine lipid nanodiscs and the kinetics of activation at 15°C and pH 8.7 was investigated by time-resolved absorption spectroscopy and global kinetic analysis. For both proteins, the kinetics in nanodiscs shows the characteristics observed in the native membranes, and is described by a multisquare model with Schiff base deprotonation at the lumirhodopsin I intermediate stage. The results indicate that the protein sequence controls the extent of Schiff base deprotonation and accumulation of intermediates, and thus plays the main role in the different activation kinetics observed between human and bovine rhodopsins. The membrane lipid does have a minor role by modulating the timing of the kinetics, with the nanodisc environment leading to an earlier Schiff base deprotonation.