Characterization of the Simultaneous Decay Kinetics of Metarhodopsin States II and III in Rhodopsin by Solution-State NMR Spectroscopy

The mammalian visual dim-light photoreceptor rhodopsin is considered a prototype Gprotein-coupled receptor. Here, we characterize the kinetics of its light-activation process. Milligram quantities of ,epsilon-N-15-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 metaII and metaIII states emerge in parallel with a relative ratio of about 3:1. Transient formation of the metaIII state was confirmed by flash photolysis experiments. From analysis of the site-resolved kinetic data we propose the involvement of the E-2-loop in the formation of the metaIII state.