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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 98; no. 9; pp. 4888 - 4892
• Main Authors: Loewen, M C, Klein-Seetharaman, J, Getmanova, E V, Reeves, P J, Schwalbe, H, Khorana, H G
• Format: Journal Article
• Language: English
• Published: 24.04.2001
• ISSN: 0027-8424
• DOI: 10.1073/pnas.051633098

super(19)F nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution super(19)F 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 super(19)F 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 degree C by solution super(19)F NMR. Distinct chemical shifts were observed for all of the rhodopsin super( 19)F 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 super(19)F- super(19)F 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 super(19)F NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.