NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: applicability of solution (19)F NMR.
We report high resolution solution (19)F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF(3)-CH(2)-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6-10 mg), in dodecylmaltoside, were analyzed at 20 degrees C by solution (19)F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts 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. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for (19)F labels at positions 67 (-0.2 ppm) and 140 (-0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution (19)F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.
Klein-Seetharaman
J
J
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Getmanova
E V
EV
Loewen
M C
MC
Reeves
P J
PJ
Khorana
H G
HG
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002461:Cell Line, Transformed; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003624:Darkness; D003902:Detergents / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D005462:Fluorine Radioisotopes; D005960:Glucosides / Q000737:chemistry; D008027:Light; D008559:Memantine / Q000031:analogs & derivatives; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D012996:Solutions
C114150:1-amino-3-fluoromethyl-5-methyladamantane; D003902:Detergents; D005462:Fluorine Radioisotopes; D005960:Glucosides; D012996:Solutions; C018619:octyl-beta-D-glucoside; C040358:dodecyl maltoside; D012243:Rhodopsin; D003545:Cysteine; D004134:Dimyristoylphosphatidylcholine; D008559:Memantine