The Energetics of Chromophore Binding in the Visual Photoreceptor Rhodopsin

Abstract

The visual photoreceptor rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that stabilizes its inverse agonist ligand, 11-cis-retinal (11CR), by a covalent, protonated Schiff base linkage. In the visual dark adaptation, the fundamental molecular event after photobleaching of rhodopsin is the recombination reaction between its apoprotein opsin and 11CR. Here we present a detailed analysis of the kinetics and thermodynamics of this reaction, also known as the “regeneration reaction”. We compared the regeneration of purified rhodopsin reconstituted into phospholipid/detergent bicelles with rhodopsin reconstituted into detergent micelles. We found that the lipid bilayer of bicelles stabilized the chromophore-free opsin over the long timescale required for the regeneration experiments, and also facilitated the ligand reuptake binding reaction. We utilized genetic code expansion and site-specific bioorthogonal labeling of rhodopsin with Alexa488 to enable, to our knowledge, a novel fluorescence resonance energy transfer-based measurement of the binding kinetics between opsin and 11CR. Based on these results, we report a complete energy diagram for the regeneration reaction of rhodopsin. We show that the dissociation reaction of rhodopsin to 11CR and opsin has a 25-pM equilibrium dissociation constant, which corresponds to only 0.3 kcal/mol stabilization compared to the noncovalent, tightly bound antagonist-GPCR complex of iodopindolol and β-adrenergic receptor. However, 11CR dissociates four orders-of-magnitude slower than iodopindolol, which corresponds to a 6-kcal/mol higher dissociation free energy barrier. We further used isothermal titration calorimetry to show that ligand binding in rhodopsin is enthalpy driven with –22 kcal/mol, which is 12 kcal/mol more stable than the antagonist-GPCR complex. Our data provide insights into the ligand-receptor binding reaction for rhodopsin in particular, and for GPCRs more broadly.