Methylation of the active-site lysine of rhodopsin.
Purified bovine rhodopsin was reductively methylated with formaldehyde and pyridine/borane with the incorporation of approximately 20 methyl groups in the protein. Rhodopsin contains 10 non-active-site lysines, which account for the uptake of the 20 methyl groups. The permethylated rhodopsin thus formed is active toward bleaching, regeneration with 11-cis-retinal, and the activation of the GTPase (G protein) when photolyzed. The critical active-site lysine of permethylated rhodopsin can be liberated by photolysis. This lysine can be reductively methylated at 4 degrees C. Methylation under these conditions leads to the incorporations of approximately 1.5 methyl groups per opsin molecule using radioactive formaldehyde, with the ratio of epsilon-dimethyllysine:epsilon-monomethyllysine:lysine being approximately 5:4:1. The modified opsin(s) can regenerate with 11-cis-retinal to produce a mixture of active-site methylated and unmethylated rhodopsins having a lambda max = 512 nm. Using [14C]formaldehyde and [3H]retinal followed by reduction of the Schiff base, digestion, and chromatography showed that the active-site N-methyllysine was bound to the retinal. Treatment of the methylated opsin mixture (containing 1.5 active-site methyl groups) with o-phthalaldehyde/mercaptoethanol to functionalize the opsin bearing unreacted lysine, followed by regeneration with 11-cis-retinal and chromatographic separation, led to the preparation of the pure active-site epsilon-lysine monomethylated rhodopsin with a lambda max = 520 nm, significantly shifted bathochromically from rhodopsin or permethylated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005557:Formaldehyde / Q000378:metabolism; D008239:Lysine; D008745:Methylation; D010786:Photoreceptor Cells / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013053:Spectrophotometry
D012168:Retinal Pigments; D005557:Formaldehyde; D012243:Rhodopsin; D008239:Lysine; D012172:Retinaldehyde