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

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Abstract
The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6- 15 N-lysine and 2- 13 C-glycine. Typical yields were 1.5–1.8 mg/liter. Incorporation of 6- 15 N-lysine was quantitative, whereas that of 2- 13 C-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 15 N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6- 15 N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 Å, 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.