Role of putative anion-binding sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin
Role of putative anion-binding sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin
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DOI：
10.1021/bi047500f
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发表时间：
2005-03-29
期刊：
影响因子：
2.9
通讯作者：
Demura, M
中科院分区：
文献类型：
--
作者：
Sato, M;Kubo, M;Demura, M
法老钠杆菌（Natronomonas pharaonis halorhodopsin, NpHR）是一种向内的光驱动Cl离子泵。为了有效的Cl-运输，假设在细胞外（EC）和细胞质（CP）通道中存在Cl-结合或相互作用位点。候选通道包括EC通道中的Arg123和Thr126以及CP通道中的Lys215和Thr218。这些氨基酸残基在阴离子结合和光循环中的作用已经通过这些位置的氨基酸残基的突变进行了研究。通过将Cl-加入到无阴离子的NpHR中，圆二色性的变化和吸收最大值的变化来分析阴离子结合。在某些EC残基被替换的突变体中，结合亲和力受到影响；这一发现揭示了Ar123的重要性。另一方面，CP通道中某些残基被取代的突变体（CP突变体）的解离常数没有变化。这些突变体的光周期也被检查，在EC突变体的情况下，过渡到最后一步被大大延迟；另一方面，在CP突变体中。除了缺乏o光中间体的K215Q外，L2光中间体的衰变时间明显延长。这些结果表明Thr218对Cl-结合到CP通道的重要性。在此基础上，讨论了NpHR可能的阴离子输运机制。
Natronomonas (Natronobacterium.) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl- ion pump. For efficient Cl- transport, the existence of Cl-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl- to anion-free NpHR. The binding affinity was affected in Mutants in which certain EC residues had been replaced; this finding revealed the importance of Ar123. On the other hand, Mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC Mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants. L2 -photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl- to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.