Changing the location of the Schiff base counterion in rhodopsin.
Rhodopsin and all of the vertebrate visual pigments have a carboxylic acid residue, Glu113, in the third transmembrane segment that serves as a counterion to the protonated Schiff base nitrogen of the chromophore. We show here that the counterion in bovine rhodopsin can be moved from position 113 to 117 without significantly changing the wild-type spectral properties of the protein. A series of double mutants were constructed where the Glu113 counterion was changed to Gln and an Asp residue was substituted for amino acid residues from position 111 to 121 in the third transmembrane segment of the protein. Only at position 117 can an Asp fully substitute for the counterion at position 113. The double mutant E113Q,-A117D has an absorption maximum at 493 nm which is independent of pH in the range 5.6-8.4 and independent of the presence of external chloride anions. An Asp at no other position tested in the third transmembrane segment can fully substitute for the Glu counterion at position 113. Partial substitution is observed for an Asp at position 120. Residues 113, 117, and 120 are expected to lie along the same face of an alpha-helix. These results suggest that the Schiff base nitrogen in rhodopsin is located between residues 113 and 117 but there is enough flexibility in the protein to allow partial interaction with an Asp at position 120. Position 117 is the same location of the counterion in the related biogenic amine receptors.
Zhukovsky
E A
EA
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Robinson
P R
PR
Oprian
D D
DD
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005813:Genes, Synthetic; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017981:Receptors, Neurotransmitter / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D012168:Retinal Pigments / Q000235:genetics; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012545:Schiff Bases; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism
D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D012168:Retinal Pigments; D012545:Schiff Bases; D012243:Rhodopsin; D015293:Transducin