Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy

Abstract

The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype G protein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of α,ε-(15)N-labeled tryptophan rhodopsin were produced in stably transfected HEK293 cells. Assignment of the chemical shifts of the indole signals was achieved by generating the single-point-tryptophan to phenylalanine mutants, and the kinetics of each of the five tryptophan residues were recorded. We find kinetic partitioning in rhodopsin decay, including three half-lives, that reveal two parallel processes subsequent to rhodopsin activation that are related to the photocycle. The meta II and meta III states emerge in parallel with a relative ratio of about 3:1. Transient formation of the meta III state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E2 -loop in the formation of the meta III state.

Keywords: G-protein coupled receptors; NMR spectroscopy; bovine rhodopsin; membrane protein dynamics; photocycles.