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

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.