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

¹⁹F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution ¹⁹F NMR spectra of fluorine-labeled rhodopsin in detergent micelles were described, demonstrating the applicability of this technique to studies of tertiary structure in the cytoplasmic domain. To quantitate tertiary contacts we have applied a transient one-dimensional difference NOE solution ¹⁹F NMR experiment to this system, permitting assessment of proximities between fluorine labels specifically incorporated into different regions of the cytoplasmic face. Three dicysteine substitution 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 and analyzed at 20°C by solution ¹⁹F NMR. Distinct chemical shifts were observed for all of the rhodopsin ¹⁹F labels in the dark. An up-field shift of the Cys-316 resonance in the Cys-65-Cys-316 mutant suggests a close proximity between the two residues. When analyzed for ¹⁹F-¹⁹F NOEs, a moderate 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, indicating proximity between these sites. No NOE enhancement was observed for the Cys-140-Cys-316 pair. These NOE effects demonstrate a solution ¹⁹F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.