Pressure‐induced Isomerization of Retinal on Bacteriorhodopsin as Disclosed by Fast Magic Angle Spinning NMR †
Pressure‐induced Isomerization of Retinal on Bacteriorhodopsin as Disclosed by Fast Magic Angle Spinning NMR †
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DOI：
10.1562/2006-06-20-rc-941
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发表时间：
2007-02
影响因子：
3.3
通讯作者：
I. Kawamura;Y. Degawa;S. Yamaguchi;K. Nishimura;S. Tuzi;H. Saitô;A. Naito
中科院分区：
文献类型：
--
作者：
I. Kawamura;Y. Degawa;S. Yamaguchi;K. Nishimura;S. Tuzi;H. Saitô;A. Naito
细菌视紫红质（bR）是盐生盐杆菌紫膜中的一种视网膜蛋白，其功能是光驱动的质子泵。我们通过分析[N-15 N]Lys标记的bR的15 N交叉极化魔角自旋（CP-MAS）NMR光谱，检测到bR中的retinal的压力诱导异构化。在15 N-NMR光谱中，在黑暗中，在4 kHz的MAS频率下，在148.0和155.0 ppm处分别在质子化席夫碱中的Lys N-异丙基中观察到全反式和13-顺式视黄醇构型。当MAS频率增加到12 kHz（对应于63 bar的样品压力）时，[N-15 N]Lys在视黄醛席夫碱中的15 N-NMR信号被加宽。另一方面，其他[N-15 N]Lys未显示增宽。随后，当MAS频率从12 kHz降低至4 kHz时，观察到155.0 ppm下13-顺式视黄醛的席夫碱中[N-15 N]Lys的信号强度增加。这些结果表明，在63巴的压力下，视网膜中[全反式-bR]/[13-顺式-bR]的平衡常数降低。压力引起的结构变化主要发生在视网膜附近。因此，在显微镜下，视网膜周围的氢键网络会被持续施加的压力破坏或扭曲。因此，清楚地表明，由快速MAS频率诱导的增加的压力在膜蛋白bR中产生了从全反式到13顺式状态的视黄醛异构化。
Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light‐driven proton pump. We have detected pressure‐induced isomerization of retinal in bR by analyzing 15N cross polarization‐magic angle spinning (CP‐MAS) NMR spectra of [ζ‐15N]Lys‐labeled bR. In the 15N‐NMR spectra, both all‐trans and 13‐cis retinal configurations have been observed in the Lys Nζ in protonated Schiff base at 148.0 and 155.0 ppm, respectively, at the MAS frequency of 4 kHz in the dark. When the MAS frequency was increased up to 12 kHz corresponding to the sample pressure of 63 bar, the 15N‐NMR signals of [ζ‐15N]Lys in Schiff base of retinal were broadened. On the other hand, other [ζ‐15N]Lys did not show broadening. Subsequently, the increased signal intensity of [ζ‐15N]Lys in Schiff base of 13‐cis retinal at 155.0 ppm was observed when the MAS frequency was decreased from 12 to 4 kHz. These results showed that the equilibrium constant of [all‐trans‐bR]/[13‐cis‐bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen‐bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all‐trans to 13‐cis state in the membrane protein bR.