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

Michèle C. Loewen, Judith Klein-Seetharaman, Elena V. Getmanova, Philip J. Reeves, Harald Schwalbe, H. Gobind Khorana

Research output: Contribution to journalArticlepeer-review

69 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4888-4892
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number9
DOIs
StatePublished - Apr 24 2001
Externally publishedYes

Keywords

  • G protein-coupled receptors
  • Membrane protein
  • NMR structure contacts
  • Signal transduction
  • Site-directed F labeling

ASJC Scopus subject areas

  • General

Fingerprint

Dive into the research topics of 'Solution <sup>19</sup>F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin'. Together they form a unique fingerprint.

Cite this