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.
Departments of Biology and Chemistry, Massachusetts Institute of Technology 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
P J
PJ
Reeves
J
J
Hwa
H G
HG
Khorana
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