[94]13C NMR spectroscopy of the chromophore of rhodopsin

Publisher Summary This chapter discusses the C nuclear magnetic resonance (nmr) spectroscopy of the chromophore of rhodopsin. C nmr spectroscopy has provided valuable information on the structure and function of water-soluble proteins. Rhodopsin is one membrane protein for which C nmr has proved feasible, as these drawbacks may be remedied by the introduction of a C enriched retinylidene chromophore and detergent solubilization. Solubilization by octyl glucoside enables high concentrations of rhodopsin to be obtained, as the lipids can be removed by chromatography and the detergent is not concentrated by ultrafiltration because of its high carboxymethyl cellulose (CMC). Octyl glucoside does not contribute resonances in the alkene region of the C nmr spectrum—that is, approximately 120–130 ppm. The rhodopsin/detergent micelle size appears to be relatively small, which leads to a short rotational correlation time and therefore narrow linewidths. The low ratio obtained for enriched rhodopsin indicates that free opsin is removed from regenerated rhodopsin during affinity chromatography, thereby maximizing the ratio.