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

摘要

We report high resolution solution 19F NMR spectra offluorine-labeled rhodopsin mutants in detergent micelles. Singlecysteine substitution mutants in the cytoplasmic face of rhodopsin werelabeled by attachment of the trifluoroethylthio (TET),CF3-CH2-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 mutantrhodopsins (6–10 mg), in dodecylmaltoside, were analyzed at 20°C bysolution 19F NMR spectroscopy. The spectrarecorded in the dark showed the following chemical shifts relative totrifluoroacetate: 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, allmutants showed chemical shifts downfield that of free TET (6.5 ppm). Onillumination to form metarhodopsin II, upfield changes in chemicalshift were observed for 19F labels at positions67 (−0.2 ppm) and 140 (−0.4 ppm) and downfield changes for positions248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change wasobserved at positions 311 and 245. On decay of metarhodopsin II, thechemical shifts reverted largely to those originally observed in thedark. The results demonstrate the applicability of solution19F NMR spectroscopy to studies of the tertiarystructures in the cytoplasmic face of intact rhodopsin in the dark andon light activation.