Smith S O, de Groot H, Gebhard R, Lugtenburg J
Department of Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Photochem Photobiol. 1992 Dec;56(6):1035-9. doi: 10.1111/j.1751-1097.1992.tb09726.x.
Magic angle spinning (MAS)13C-NMR spectra of the metarhodopsin II intermediate have been obtained using bovine rhodopsin regenerated with retinal 13C-labeled at the C-13 and C-15 positions to investigate the protonation state of the retinal Schiff base linkage. The 13C-labeled rhodopsin was reconstituted into 1,2-dipalmitoleoylphosphatidylcholine bilayers to increase the amount of meta II trapped at low temperature. Both the 13C-15 (159.2 ppm) and 13C-13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the 13C-15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70-90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules out the possibility that meta II non-covalently binds retinal or is a carbinolamine intermediate and provides convincing evidence that Schiff base deprotonation occurs in the meta I-meta II transition, an event that is likely to be important in triggering the activation of transducin.
利用在C-13和C-15位置用13C标记的视黄醛再生的牛视紫红质，获得了变视紫红质II中间体的魔角旋转（MAS）13C-NMR光谱，以研究视黄醛席夫碱连接的质子化状态。将13C标记的视紫红质重组到1,2-二棕榈酰油酰磷脂酰胆碱双层中，以增加在低温下捕获的变视紫红质II的量。13C-15（159.2 ppm）和13C-13（144.0 ppm）的各向同性化学位移都是未质子化席夫碱的特征，而13C-15位移与视黄醛（191 ppm）或先前提出的作为变视紫红质II阶段席夫碱水解中间体的四面体甲醇胺基团（70-90 ppm）的位移有显著差异。这排除了变视紫红质II与视黄醛非共价结合或为甲醇胺中间体的可能性，并提供了令人信服的证据，表明席夫碱去质子化发生在变视紫红质I-变视紫红质II转变过程中，这一事件可能在触发转导素激活中起重要作用。
