Structure and function in rhodop binding to purified opsin mutant, detergent mixtures serve as prob (G-protein-coupled receptors/signal transduction/11-cis-retinal/

In the current standard procedure for prep- aration of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 JuM 11-cis-retinal before detergent solubilization for purification. We found that binding of 11-cis-retinal to opsin mutants with single amino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (A164V) was far from complete and required much higher concentrations of 11-cis-retinal. By isolation of the expressed opsins in a stable form, kinetic studies of retinal binding to the opsins in vitro have been carried out by using defined phospholipid-detergent mix- tures. The results show wide variation in the rates of 11-cis- retinal binding. Thus, the in vitro reconstitution procedure serves as a probe of the retinal-binding pocket in the opsins. Further, a method is described for purification and charac- terization of the rhodopsin mutants after retinal binding to the opsins in vitro.