Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin:: Expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line
Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin:: Expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line
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DOI：
10.1073/pnas.96.2.487
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发表时间：
1999-01-19
影响因子：
11.1
通讯作者：
Smith, SO
中科院分区：
文献类型：
--
作者：
Eilers, M;Reeves, PJ;Smith, SO
利用HEK293S细胞悬浮培养，在含有6-N-15-赖氨酸和2-C-13-甘氨酸的特定培养基中表达了与哺乳动物光感受器视紫红质相对应的载脂蛋白。典型的产量为1.5-1.8毫克/升。6-N-15-赖氨酸的掺入是定量的，而2-C-13-甘氨酸的掺入约为60%。11-顺式视网膜结合形成的视紫红质色素与天然牛视紫红质在光谱上没有区别，标记的视紫红质与脂质体结合后获得了魔角旋转(MAS)核磁共振谱。在6-N-15-赖氨酸标记的视紫红质的分子吸收光谱中，在156.8 ppm处观察到与质子化的视黄基席夫碱氮相对应的N-15共振，这种化学位移对应于有效的席夫碱-反离子距离大于4埃，与席夫碱氮和谷氨酸-113型反离子氢键结合位置上的结构水一致。本研究表明，用MAS核磁共振技术研究视紫红质和其他G蛋白偶联受体的结构是可行的。
The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-N-15-lysine and 2-C-13-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-N-15-lysine was quantitative, whereas that of 2-C-13-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin, Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The N-15 resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-N-15-lysine-labeled rhodopsin, This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 Angstrom, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.