Participation of the BC Loop in the Correct Folding of Bacteriorhodopsin as Revealed by Solid-state NMR
Participation of the BC Loop in the Correct Folding of Bacteriorhodopsin as Revealed by Solid-state NMR
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DOI：
10.1111/j.1751-1097.2009.00536.x
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发表时间：
2009-03-01
影响因子：
3.3
通讯作者：
Naito, Akira
中科院分区：
文献类型：
--
作者：
Kawamura, Izuru;Tanabe, Junko;Naito, Akira
通过观察 [1-C-13]Val 和 [N-15]Pro 标记的 bR 的 C-13 和 N-15 固态 NMR 光谱，研究了两种不同的视网膜重建过程中细菌视紫红质 (bR) 的结构变化。我们发现 BC 环的 NMR 信号对蛋白质结构和动力学的变化敏感，从野生型 (WT) bR 到细菌视蛋白 (bO)、再生 bR 和 E1001 bR。将视黄醛添加到从光漂白的 WT-bR 获得的 bO 中后制备再生 bR。 E1001 bR 是在向生长的细胞中添加视网膜后，从称为 E1001 的视网膜缺陷菌株中培养出来的。在 WT-bR BC 环中 Pro70 处的 N-15 NMR 信号在 122.4 p.p.m. 处观察到，而在 bO 和再生 bR 中信号分别不明显或部分受到抑制。同样，BC 环路中 Val69 处的 13 C NMR 信号位于 172.0 p.p.m。在 WT-bR 中观察到的再生 bR 和 bO 均显着降低。这些结果表明，在去除视网膜后，bO 中 BC 环的动态结构发生了显着改变。结果，视网膜向bO的再生过程未能恢复正确的蛋白质结构。另一方面，E1001 bR中BC环处的C-13和N-15 NMR信号出现在与WT-bR相同的位置。目前的研究结果表明，BC环在再生bR中可能并不总是正确折叠，这导致再生bR与WT-bR相比具有不同的特性。
Structural changes in bacteriorhodopsin (bR) in two different processes of retinal reconstitutions were investigated by observing the C-13 and N-15 solid-state NMR spectra of [1-C-13]Val- and [N-15]Pro-labeled bR. We found that NMR signals of the BC loop were sensitive to changes in protein structure and dynamics, from wild-type (WT) bR to bacterio-opsin (bO), regenerated bR and E1001 bR. Regenerated bR was prepared following the addition of retinal into bO obtained from photobleached WT-bR. E1001 bR was cultured from a retinal-deficient strain termed E1001 following the addition of retinal to growing cells. N-15 NMR signal at Pro70 in the BC loop in WT-bR was observed at 122.4 p.p.m., whereas signals were not apparent or partly suppressed in bO and regenerated bR, respectively. Similarly, the 13 C NMR signal at Val69 in the BC loop at 172.0 p.p.m. that was observed in WT-bR was significantly decreased in both regenerated bR and bO. These results suggest that the dynamic structure of the BC loop in bO was substantially altered following the removal of retinal. As a consequence, the correct protein structure failed to be recovered via the regenerating process of retinal to bO. On the other hand, C-13 and N-15 NMR signals at the BC loop in E1001 bR appeared at positions identical to those of WT-bR. The results of the current study indicate that the BC loop may not always fold correctly in the regenerated bR, which leads to different properties in the regenerated bR compared to that of WT-bR.