NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin:: Applicability of solution 19F NMR
NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin:: Applicability of solution 19F NMR
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DOI：
10.1073/pnas.96.24.13744
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发表时间：
1999-11-23
影响因子：
11.1
通讯作者：
Khorana, HG
中科院分区：
文献类型：
--
作者：
Klein-Seetharaman, J;Getmanova, EV;Khorana, HG
我们报告高分辨率溶液F-19 NMR光谱的氟标记的视紫红质突变体在洗涤剂胶束。通过二硫键连接三氟乙硫基（泰特），CF 3-CH 2-S，标记视紫红质胞质面中的单半胱氨酸取代突变体。因此制备了视紫红质中氨基酸位置67、140、245、248、311和316处的TET标记的半胱氨酸突变体。在20 ℃下通过溶液F-19 NMR光谱分析十二烷基麦芽糖苷中的纯化的突变视紫红质（6-10 mg）。在黑暗中记录的光谱显示以下相对于三氟乙酸盐的化学位移：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;和Cys-316。10.0 ppm.因此，所有突变体均显示游离泰特（6.5 ppm）的化学位移低场。在照射以形成后视紫红质II时，观察到位置67（-0.2ppm）和140（-0.4ppm）处的F-19标记物的化学位移的高场变化以及位置248（+0.1ppm）和316（+0.1ppm）处的低场变化，而在位置311和245处观察到很少或没有变化。在变视紫红质II的衰变过程中，化学位移基本上恢复到最初在黑暗中观察到的水平。结果表明，适用于解决F-19核磁共振光谱的三级结构的研究在细胞质面的完整的视紫红质在黑暗中和战斗激活。
We report high resolution solution F-19 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), 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 mutant rhodopsins (6-10 mg), in dodecylmaltoside, were analyzed at 20 degrees C by solution F-19 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 F-19 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 F-19 NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on fight activation.