Rhodopsin is the major membrane protein of the rod outer segment (ROS), a highly ordered array of hundreds of discs that are enclosed by a separate plasma membrane, in vertebrate retina. As a visual photoreceptor it contains a photoreactive retinal chromophore that is covalently attached to the protein and is converted into a receptor agonist by light. Rhodopsin is the best studied GPCR; structural studies, however, yielding information about its activated state shall now be initiated using light-triggered time-resolved NMR spectroscopy. We have obtained both selectively and uniformly isotope-labeled samples of rhodopsin in a high level mammalian expression system (HEK293) of the bovine opsin gene yielding mg quantities of functional protein with all posttranslational modifications for NMR studies. We will now prepare opsin using the cell-free expression system in collaboration with P2 (Dötsch / Bernhard) and compare functionality and NMR spectra. We will also study the interaction of rhodopsin with arrestin and peptides derived from transducin to obtain information about protein-protein interactions involved in the signalling cascade. Light-triggered time-resolved NMR spectroscopy will be extended to study ATP turnover and substrate stoichiometry for a full length ABC transporter together with P6 (Glaubitz).

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