The Energetics of the Chromophore Regeneration Pathway in Rhodopsin

Abstract

The regeneration of rhodopsin after photobleaching (i.e., the binding of 11-cis-retinal) is a key molecular event responsible for dark adaptaion. Here we present a detailed study on the kinetics and thermodynamics of this reaction. We utilized a phospholipid/detergent bicelle system that provided superior opsin thermostability compared with other reconstitution systems. We employed recombinant rhodopsin with a genetically-encoded azido-Phe (azF) [1] that was subsequently modified with Alexa488 fluorophore in a stoichiometric bioorthogonal labeling reaction[2]. The Alexa488-Rho allowed us to develop FRET-based assays to monitor retinal entry into the binding pocket and the subsequent formation of Schiff base bond. We also showed that the diffusion of 11-cis-retinal among the bicelles was not rate-limiting and that the recombination reaction followed a simple second-order rate law. We measured the kinetics of the recombination reaction at different temperatures to assess the enthalpic and entropic contribution of the regeneration reaction. We further used isothermal titration calorimetry to obtain the overall change in enthalpy. To study the reverse reaction (i.e., the dissociation of retinal from opsin) we performed a chromophore exchange experiment in which we observed very slow (∼10-7 s-1) exchange of 11-cis-retinal for 9-cis-retinal in rhodopsin. To our knowledge, this is the first in vitro experimental demonstration of the chromophore exchange reaction in rhodopsin. Based on the overall results of the study, we derived an energy diagram for the rhodopsin regeneration reaction.[1] Huber, T. & Sakmar, T. P. (2014) Chemical Biology Methods for Investigating G Protein-Coupled Receptor Signaling. Chem. Biol.[2]Tian, H., Naganathan, S., Kazmi, M. A., Schwartz, T. W., Sakmar, T. P. and Huber, T. (2014), Bioorthogonal Fluorescent Labeling of Functional G-Protein-Coupled Receptors. ChemBioChem.