Structure and function in rhodopsin: asymmetric reconstitution of rhodopsin in liposomes.
We report on preparation of rhodopsin proteoliposomes with the cytoplasmic domain of rhodopsin facing the exterior of the proteoliposomes. Rhodopsin purified from rod outer segments of bovine retinae by immunoaffinity chromatography in octyl glucoside was reconstituted into liposomes prepared from soybean phospholipids by detergent dialysis. The orientation of rhodopsin in the liposomes was determined by susceptibility of its C terminus to papain and the endoproteinase, Asp-N, followed by SDS/PAGE, which showed that the cytoplasmic domain in at least 90% of rhodopsin faced the exterior of the proteoliposomes. By using escape of (32)P-KP(i) encapsulated in the proteoliposomes as the assay, the half-life of the proteasomes was approximately 8 days. After light activation, rhodopsin in proteoliposomes showed the rate of decay of metarhodopsin II and the initial rate of transducin activation comparable with the rates of rhodopsin in rod outer segment membranes. This finding demonstrates the functional capability of rhodopsin in proteoliposomes for kinetic studies of protein-protein interactions.
Niu
Li
L
Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Kim
Jong-Myoung
JM
Khorana
H Gobind
HG
D000818:Animals; D002417:Cattle; D004355:Drug Stability; D010450:Endopeptidases; D066298:In Vitro Techniques; D007700:Kinetics; D008027:Light; D008666:Metalloendopeptidases; D010206:Papain; D011510:Proteolipids; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects
D011510:Proteolipids; C020485:proteoliposomes; C013110:metarhodopsins; D012243:Rhodopsin; D010450:Endopeptidases; D010206:Papain; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N