Opsin/all-trans-retinal complex activates transducin by different mechanisms than photolyzed rhodopsin.
In rhodopsin, the 11-cis-retinal chromophore forms a complex with Lys296 of opsin via a protonated Schiff base. Absorption of light initiates the activation of rhodopsin by cis/trans photoisomerization of retinal. Thermal relaxation through different intermediates leads into the metarhodopsin states which bind and activate transducin (Gt) and rhodopsin kinase (RK). all-trans-Retinal also recombines with opsin independent of light, forming activating species of the receptor. In this study, we examined the mechanism by which all-trans-retinal activates opsin. To exclude other amines except active site Lys296 from formation of Schiff bases, we reductively methylated rhodopsin (PM-rhodopsin), which we then bleached to generate PM-opsin. Using spectroscopic methods and a Gt activation assay, we found that all-trans-retinal interacted with PM-opsin, producing a noncovalent complex that activated Gt. The residual nucleotide exchange in Gt catalyzed by opsin was approximately 1/250 lower relative to that of photoactivated rhodopsin (pH 8.0, 23 degrees C). Addition of equimolar all-trans-retinal led to an occupancy of one-tenth of the putative retinal binding site(s) of opsin and enhanced the Gt activation rate 2-fold. When the concentration of all-trans-retinal was increased to saturation, the Gt activation rate of the opsin/all-trans-retinal complex was approximately 1/33 lower compared to that of photoactivated rhodopsin. We conclude that all-trans-retinal can form a noncovalent complex with opsin that activates Gt by different mechanisms than photolyzed rhodopsin.
Jäger
S
S
Institut für Medizinische Physik und Biophysik, Charité, Humboldt-Universität zu Berlin, Ziegelstrasse 5-9, Germany.
Palczewski
K
K
Hofmann
K P
KP
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003624:Darkness; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008239:Lysine; D008745:Methylation; D010782:Photolysis; D012172:Retinaldehyde / Q000494:pharmacology*; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D017299:Rod Opsins / Q000378:metabolism*; D012545:Schiff Bases; D013053:Spectrophotometry; D013237:Stereoisomerism; D015293:Transducin / Q000378:metabolism*
D017299:Rod Opsins; D012545:Schiff Bases; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D015293:Transducin; D008239:Lysine; D012172:Retinaldehyde