期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 98, 期 9, 页码 4888-4892出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.051633098
关键词
G protein-coupled receptors; signal transduction; site-directed F-19 labeling; NMR structure contacts; membrane protein
资金
- NEI NIH HHS [R01 EY011716, EY11716] Funding Source: Medline
- NIGMS NIH HHS [GM28289, R01 GM028289] Funding Source: Medline
F-19 nuclear Overhauser effects (NOEs) between fluorine labels on the cytoplasmic domain of rhodopsin solubilized in detergent micelles are reported. Previously, high-resolution solution F-19 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 F-19 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 degreesC by solution F-19 NMR. Distinct chemical shifts were 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 close proximity between the two residues. When analyzed for F-19-F-19 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 F-19 NMR method for analysis of tertiary contacts in high molecular weight proteins, including membrane proteins.
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