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Angew Chem Int Ed Engl. 2014 Feb 17;53(8):2078-84. doi: 10.1002/anie.201309581. Epub 2014 Feb 6.

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

Author information

1
Institute for Organic Chemistry and Chemical Biology, Center of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany).

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

PMID:
24505031
DOI:
10.1002/anie.201309581
[Indexed for MEDLINE]

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