Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
Biophys J. 2012 Jun 20;102(12):2906-15. doi: 10.1016/j.bpj.2012.05.008. Epub 2012 Jun 19.
Halorhodopsin from NpHR is a light-driven Cl(-) pump that forms a trimeric NpHR-bacterioruberin complex in the native membrane. In the case of NpHR expressed in Escherichia coli cell, NpHR forms a robust homotrimer in a detergent DDM solution. To identify the important residue for the homotrimer formation, we carried out mutation experiments on the aromatic amino acids expected to be located at the molecular interface. The results revealed that Phe(150) was essential to form and stabilize the NpHR trimer in the DDM solution. Further analyses for examining the structural significance of Phe(150) showed the dissociation of the trimer in F150A (dimer) and F150W (monomer) mutants. Only the F150Y mutant exhibited dissociation into monomers in an ionic strength-dependent manner. These results indicated that spatial positions and interactions between F150-aromatic side chains were crucial to homotrimer stabilization. This finding was supported by QM calculations. In a functional respect, differences in the reaction property in the ground and photoexcited states were revealed. The analysis of photointermediates revealed a decrease in the accumulation of O, which is important for Cl(-) release, and the acceleration of the decay rate in L1 and L2, which are involved in Cl(-) transfer inside the molecule, in the trimer-dissociated mutants. Interestingly, the affinity of them to Cl(-) in the photoexcited state increased rather than the trimer, whereas that in the ground state was almost the same without relation to the oligomeric state. It was also observed that the efficient recovery of the photocycle to the ground state was inhibited in the mutants. In addition, a branched pathway that was not included in Cl(-) transportation was predicted. These results suggest that the trimer assembly may contribute to the regulation of the dynamics in the excited state of NpHR.
嗜盐菌视紫红质（NpHR）是一种光驱动的氯离子泵，在天然膜中形成三聚体 NpHR-细菌视紫红质复合物。在大肠杆菌细胞中表达的 NpHR 的情况下，NpHR 在去污剂 DDM 溶液中形成稳定的同源三聚体。为了鉴定同源三聚体形成的重要残基，我们对预期位于分子界面的芳香族氨基酸进行了突变实验。结果表明，苯丙氨酸（150）对于在 DDM 溶液中形成和稳定 NpHR 三聚体是必不可少的。进一步分析检查苯丙氨酸（150）的结构意义表明，在 F150A（二聚体）和 F150W（单体）突变体中三聚体解离。只有 F150Y 突变体表现出在离子强度依赖性下的单体解离。这些结果表明，F150-芳香族侧链的空间位置和相互作用对于同源三聚体的稳定至关重要。这一发现得到了 QM 计算的支持。在功能方面，揭示了在基态和光激发态下反应性质的差异。光中间体分析表明，对于氯离子释放很重要的 O 的积累减少，以及涉及分子内氯离子转移的 L1 和 L2 的衰减速率加快，在三聚体解离突变体中。有趣的是，与三聚体相比，它们在光激发态下对氯离子的亲和力增加，而在基态下几乎相同，与寡聚态无关。还观察到突变体中光循环到基态的有效恢复受到抑制。此外，预测了一种不包括氯离子运输的分支途径。这些结果表明，三聚体组装可能有助于调节 NpHR 激发态的动力学。
