Characterization of photo-intermediates in the photo-reaction pathways of a bacteriorhodopsin Y185F mutant using in situ photo-irradiation solid-state NMR spectroscopy
Characterization of photo-intermediates in the photo-reaction pathways of a bacteriorhodopsin Y185F mutant using in situ photo-irradiation solid-state NMR spectroscopy
复制标题
使用原位光照射固态核磁共振波谱表征细菌视紫红质 Y185F 突变体的光反应途径中的光中间体
DOI：
10.1039/c5pp00154d
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Akira Naito
中科院分区：
文献类型：
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作者：
Kyosuke Oshima;Aris Shigeta;Yoshiteru Makino;Izuru Kawamura;Takashi Okitsu;Akimori Wada;Satoru Tuzi;Tetsuo Iwasa;Akira Naito
利用固体核磁共振仪研究了细菌视紫红质Y185F突变株的光反应途径。在−40°C的黑暗(D1)、520 nm光照(L1)、随后的黑暗(D2)和520 nm光照(L2)下，记录了13C CP MAS核磁共振波谱。在从D1到L1的过程中，13-顺，15-syn(CS；bR548)状态转变为CS*-(13-顺，15-syn)中间体，该中间体在−40℃下高度稳定，并且全反式(AT；bR568)状态转变为N-中间体。在D2条件下，N-中间体转变为O-中间体，在−40°C黑暗中非常稳定。在随后的520 nm光(L2)照射下，O-中间体通过AT态转变为N-中间体，而CS*-中间体没有变化。在温度升高到−20℃后，CS*-中间体转变为AT状态(或O-中间体)，随后温度升高到20℃，AT状态(或O-中间体)转变为CS状态，直到达到平衡。在本实验中，[20-13C，14-13C]视网膜的化学位移值分别提供了13C=C和15C=N构型。根据这些数据，AT和CS状态以及CS*-、N-和O-中间体的构型被确定为(13-反式，15-抗)、(13-顺，15-顺)、(13-顺，15-顺)、(13-顺，15-反)和(13-反，15-反)，Y185F-BR突变体的CS~*和O~-中间体的~(13)C核磁共振信号首次被现场光照固体核磁共振谱观测到，CS~*~-中间体的构型与CS态的构型有明显的扭曲，尽管这两个构型都被指定为(13-顺式，15-SYN)构型。
Photo-reaction pathways of a bacteriorhodopsin Y185F mutant were examined usingin situphoto-irradiation solid-state NMR spectroscopy.13C CP MAS NMR spectra were recorded at −40 °C in the dark (D1), under irradiation with 520 nm light (L1), subsequently in the dark (D2), and again under irradiation with 520 nm light (L2). In the process from D1 to L1, the 13-cis, 15-syn(CS; bR548) state changed to a CS*- (13-cis, 15-syn) intermediate, which was highly stable at −40 °C, and the all-trans(AT; bR568) state transformed to an N-intermediate. Under the D2 conditions, the N-intermediate transformed to an O-intermediate, which was highly stable at −40 °C in the dark. During subsequent irradiation with 520 nm light (L2), the O-intermediate transformed to the N-intermediate through the AT state, whereas the CS*-intermediate did not change. The CS*-intermediate was converted to the AT state (or O-intermediate) after the temperature was increased to −20 °C. Upon subsequent increase of the temperature to 20 °C, the AT state (or O-intermediate) was converted to the CS state until reaching equilibrium. In this experiment, the chemical shift values of [20-13C, 14-13C]retinal provided the 13C=C and 15C=N configurations, respectively. From these data, the configurations of the AT and CS states and the CS*-, N-, and O-intermediates were determined to be (13-trans, 15-anti), (13-cis, 15-syn), (13-cis, 15-syn), (13-cis, 15-anti), and (13-trans, 15-anti), respectively.13C NMR signals of the CS*- and O-intermediates were observed for the first time for the Y185F bR mutant byin situphoto-irradiation solid-state NMR spectroscopy and the configuration of the CS*-intermediate was revealed to be significantly twisted from that of the CS state although both were assigned as (13-cis, 15-syn) configurations.