Structure and function in rhodopsin: kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serve as probes of the retinal binding pocket.
In the current standard procedure for preparation of mammalian rhodopsin mutants, transfected COS-1 cells expressing the mutant opsin genes are treated with 5 microM 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 mixtures. 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 characterization of the rhodopsin mutants after retinal binding to the opsins in vitro.
Reeves
P J
PJ
Departments of Biology and Chemistry, Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Hwa
J
J
Khorana
H G
HG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002417:Cattle; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism*; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins; D017299:Rod Opsins; C013110:metarhodopsins; D012243:Rhodopsin; D012172:Retinaldehyde