NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: Applicability of solution F-19 NMR

Citation
J. Klein-seetharaman et al., NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: Applicability of solution F-19 NMR, P NAS US, 96(24), 1999, pp. 13744-13749
Citations number
42
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
00278424 → ACNP
Volume
96
Issue
24
Year of publication
1999
Pages
13744 - 13749
Database
ISI
SICI code
0027-8424(19991123)96:24<13744:NSISOL>2.0.ZU;2-Z
Abstract
We report high resolution solution F-19 NMR spectra of fluorine-labeled rho dopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trif luoroethylthio (TET), CF3-CH2-S, group through a disulfide linkage. TET-lab eled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 3 16 in rhodopsin were thus prepared. Purified mutant rhodopsins (6-10 mg), i n dodecylmaltoside, were analyzed at 20 degrees C by solution F-19 NMR spec troscopy. The spectra recorded in the dark showed the following chemical sh ifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys- 245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316. 10.0 ppm. Th us, all mutants showed chemical shifts downfield that of free TET (6.5 ppm) . On illumination to form metarhodopsin II, upfield changes in chemical shi ft were observed for F-19 labels at positions 67 (-0.2 ppm) and 140 (-0.4 p pm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) w hereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those original ly observed in the dark. The results demonstrate the applicability of solut ion F-19 NMR spectroscopy to studies of the tertiary structures in the cyto plasmic face of intact rhodopsin in the dark and on fight activation.