Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin:: Expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-N-15-lysine and 2-C-13-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-N-15-lysine was quantitative, whereas that of 2-C-13-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin, Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The N-15 resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-N-15-lysine-labeled rhodopsin, This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 Angstrom, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.