Chemical modification of rhodopsin and its effect on regeneration and G protein activation.
The studies reported are concerned with the functional consequences of the chemical modifications of the lysines and carboxyl-containing amino acids of bovine rhodopsin. The 10 non-active-site lysine residues of rhodopsin can be completely dimethylated and partially acetimidated (8-9 residues) with no loss in the ability of the proteins to activate the G protein when photolyzed or to regenerate with 11-cis-retinal. These modifications do not alter the net charge on the protein. Surprisingly, heavy acetylation of these lysines (eight to nine residues) with acetic anhydride, which neutralizes the positive charges of the lysine residues, yields a modified rhodopsin fully capable of activating the G protein and being regenerated. It is concluded that the non-active-site lysine residues of rhodopsin are not importantly and directly involved in interactions with the G protein during photolysis. However, this is not to say that they are unimportant in maintaining the tertiary structure of the protein because heavy modification of these residues by succinylation and trinitrophenylation produces proteins incapable of G protein activation, although the succinylated protein still regenerated. The active-site lysine of rhodopsin was readily modified and prevented from regenerating with 11-cis-retinal and with o-salicylaldehyde and o-phthalaldehyde/mercaptoethanol, two sterically similar aromatic aldehyde containing reagents which react by entirely different mechanisms. It is suggested that rhodopsin contains an aromatic binding site within its active-site region. Monoethylation, but not monomethylation, of the active-site lysine also prevented regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D005136:Eye Proteins / Q000378:metabolism*; D005557:Formaldehyde / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins; D007700:Kinetics; D010786:Photoreceptor Cells / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D015293:Transducin
D002250:Carbon Radioisotopes; D005136:Eye Proteins; C034809:GNAT1 protein, human; D012168:Retinal Pigments; D005557:Formaldehyde; D012243:Rhodopsin; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin