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
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.