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 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-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 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 A, 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.
Eilers
M
M
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.
Reeves
P J
PJ
Ying
W
W
Khorana
H G
HG
Smith
S O
SO
D000595:Amino Acid Sequence; D000818:Animals; D002247:Carbon Isotopes / Q000032:analysis; D002417:Cattle; D002460:Cell Line; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009587:Nitrogen Isotopes / Q000032:analysis; D017433:Protein Structure, Secondary; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry*; D017299:Rod Opsins / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry*; D013053:Spectrophotometry; D014867:Water / Q000737:chemistry
D002247:Carbon Isotopes; D008081:Liposomes; D009587:Nitrogen Isotopes; D017299:Rod Opsins; D012545:Schiff Bases; D014867:Water; D012243:Rhodopsin; D008239:Lysine; D012172:Retinaldehyde