Kawamura Izuru, Degawa Yoshiaki, Yamaguchi Satoru, Nishimura Katsuyuki, Tuzi Satoru, Saitô Hazime, Naito Akira
Graduate School of Engineering, Yokohama National University, Hodogaya-ku, Yokohama, Japan.
Photochem Photobiol. 2007 Mar-Apr;83(2):346-50. doi: 10.1562/2006-06-20-RC-941.
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 [zeta-15N]Lys-labeled bR. In the 15N-NMR spectra, both all-trans and 13-cis retinal configurations have been observed in the Lys N(zeta) 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 [zeta-15N]Lys in Schiff base of retinal were broadened. On the other hand, other [zeta-15N]Lys did not show broadening. Subsequently, the increased signal intensity of [zeta-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.
细菌视紫红质（bR）是嗜盐菌紫膜中的一种视黄醛蛋白，其功能是作为光驱动质子泵。我们通过分析[ζ-15N]赖氨酸标记的bR的15N交叉极化-魔角旋转（CP-MAS）核磁共振谱，检测了bR中视黄醛的压力诱导异构化。在15N核磁共振谱中，在黑暗中4 kHz的魔角旋转频率下，全反式和13-顺式视黄醛构型分别在质子化席夫碱中赖氨酸的N(ζ)处148.0 ppm和155.0 ppm被观察到。当魔角旋转频率增加到12 kHz（对应于63 bar的样品压力）时，视黄醛席夫碱中[ζ-15N]赖氨酸的15N核磁共振信号变宽。另一方面，其他[ζ-15N]赖氨酸没有显示变宽。随后，当魔角旋转频率从12 kHz降低到4 kHz时，观察到155.0 ppm处13-顺式视黄醛席夫碱中[ζ-15N]赖氨酸的信号强度增加。这些结果表明，63 bar的压力使视黄醛中[全反式-bR]/[13-顺式-bR]的平衡常数降低。还揭示了压力诱导的结构变化发生在视黄醛附近。因此，从微观上看，持续施加的压力会破坏或扭曲视黄醛周围的氢键网络。因此，清楚地证明了快速魔角旋转频率产生的压力增加导致膜蛋白bR中视黄醛从全反式异构化为13-顺式状态。
