Opsin is present as dimers in COS1 cells: identification of amino acids at the dimeric interface.
Rhodopsin in the disk membranes of rod outer segments serves as the dim-light photoreceptor and is a prototypic member of a G protein-coupled receptor family. Electron and atomic-force microscopy indicate that rhodopsin is present as dimers in the native membranes. Here, we have expressed the protein, opsin, in COS1 cells and have studied its molecular state by using FRET and by intermolecular cross-linking after site-directed cysteine mutagenesis. To observe FRET, the ends of the genes corresponding to the N termini of the cyan or yellow fluorescent proteins were fused to the ends of the genes corresponding to the C terminus of the opsin and the resulting fused genes were expressed in COS1 cells. The emission spectra in situ of the expressed proteins were recorded, and FRET was then calculated. The result indicated intermolecular interaction between opsin molecules in COS1 cells. To identify the amino acids involved in the interaction, those predicted by molecular modeling to be at the dimer interface were mutated one at a time to cysteine, and dimer formation was measured by the rate of disulfide bond formation in the presence of cupric orthophenanthroline. The mutants W175C and Y206C formed the dimers most rapidly, showing that the two amino acids were at the dimer interface.
Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Parvathi
P
Kota
Philip J
PJ
Reeves
Uttam L
UL
Rajbhandary
H Gobind
HG
Khorana
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010618:Phenanthrolines / Q000494:pharmacology; D020836:Protein Structure, Quaternary; D017299:Rod Opsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D003432:Cross-Linking Reagents; D010618:Phenanthrolines; D017299:Rod Opsins; D003545:Cysteine; C025205:1,10-phenanthroline