Solution F-19 nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

F-19 nuclear Overhauser effects (NOEs) between fluorine labels on the cytop lasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution F-19 NMR spectra of fluorine-labeled r hodopsin in detergent micelles were described, demonstrating the applicabil ity of this technique to studies of tertiary structure in the cytoplasmic d omain. To quantitate tertiary contacts we have applied a transient one-dime nsional difference NOE solution F-19 NMR experiment to this system, permitt ing assessment of proximities between fluorine labels specifically incorpor ated into different regions of the cytoplasmic face. Three dicysteine subst itution mutants (Cys-140-Cys-316, Cys-65-Cys-316. and Cys-139-Cys-251) were labeled by attachment of the trifluoroethylthio group through a disulfide linkage. Each mutant rhodopsin was prepared (8-10 mg) in dodecylmaltoside a nd analyzed at 20 degreesC by solution F-19 NMR. Distinct chemical shifts w ere observed for all of the rhodopsin F-19 labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65-Cys-316 mutant suggests a clos e proximity between the two residues. When analyzed for F-19-F-19 NOEs, a m oderate negative enhancement was observed for the Cys-65-Cys-316 pair and a strong negative enhancement was observed for the Cys-139-Cys-251 pair, ind icating proximity between these sites. No NOE enhancement was observed for the Cys-140-Cys-316 pair. These NOE effects demonstrate a solution F-19 NMR method for analysis of tertiary contacts in high molecular weight proteins , including membrane proteins.