Structure and function in rhodopsin: Kinetic studies of retinal binding topurified opsin mutants in defined phospholipid-detergent mixtures serve asprobes of the retinal binding pocket

In the current standard procedure for preparation of mammalian rhodopsin mu tants, transfected COS-1 cells expressing the mutant opsin genes are treate d with 5 mu M 11-cis-retinal before detergent solubilization for purificati on. We found that binding of 11-cis-retinal to opsin mutants with single am ino 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 mixtures. The results show wide varia tion in the rates of 11-cis-retinal binding. Thus, the in vitro reconstitut ion procedure serves as a probe of the retinal-binding pocket in the opsins . Further, a method is described for purification and characterization of t he rhodopsin mutants after retinal binding to the opsins in vitro.