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Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy
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Stehle, Jochen, Silvers, Robert, Werner, Karla, Chatterjee, Deep, Gande, Santosh, Scholz, Frank, Dutta, Arpana, Wachtveitl, Josef, Klein-Seetharaman, Judith and Schwalbe, Harald (2014) Characterization of the simultaneous decay kinetics of metarhodopsin states II and III in rhodopsin by solution-state NMR spectroscopy. Angewandte Chemie International Edition, Volume 53 (Number 8). pp. 2078-2084. doi:10.1002/anie.201309581 ISSN 1433-7851.
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Official URL: http://dx.doi.org/10.1002/anie.201309581
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-15N-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.
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