Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodiscs Using Native Lipids
Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodiscs Using Native Lipids
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使用天然脂质重组为纳米圆盘的盐视紫红质的光谱表征
DOI：
10.1016/j.bpj.2020.04.021
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
K.
中科院分区：
文献类型：
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作者：
Yamamoto;A.;Tsukamoto;T.;Suzuki;K.;Hashimoto;E.;Kobashigawa;Y.;Shibasaki;K.;Uchida;T.;Inagaki;F.;Demura;M.;Ishimori;K.
我们使用天然古菌脂质 (NL) 和具有两性离子头基的人工脂质 1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱 (POPC)，成功地将单个 Natronomonas pharaonishalorhodopsin (NpHR) 三聚体重组为纳米盘 (ND)。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳、尺寸排阻色谱法和可见圆二色光谱证实了单一三聚体 NpHR 并入 ND。通过吸收光谱检查使用 NL (NL-ND NpHR) 或 POPC (POPC-ND NpHR) 的 ND 中 NpHR 的 Cl− 结合亲和力，结果表明这些 ND 重建的 NpHR 的 Cl− 释放亲和力 (Kd,N↔O) 比从 NpHR 过表达古菌菌株 (MF) 收获的天然 NpHR 膜片段 (MF) 获得的亲和力高 10 倍以上NpHR）。这些 ND 重建的 NpHR 的光反应动力学表明，Cl− 吸收速度比 MF NpHR 更快。 ND 重建的 NpHR 和 MF NpHR 的 Cl−释放和吸收特性的这些差异可能是由于 ND 重建、三聚体状态的构象扰动以及三聚体-三聚体相互作用的丧失引起的与三聚体 NpHR 的 Cl−释放相关的蛋白质构象变化的抑制所致。另一方面，与 NL-ND NpHR 相比，POPC-ND NpHR 表现出加速的 Cl−吸收，表明古菌膜表面的负电荷调节 NpHR 的光循环。尽管NL-ND NpHR和MF NpHR嵌入相同的脂质中，但NL-ND NpHR在初始状态(Kd,initial)时观察到较低的Cl−结合亲和力，并且从NpHR'状态恢复到光反应循环的原始状态更快，这可能是因为与天然膜中的发色团、重构的ND中的细菌红素的相互作用不足。我们的结果表明，NpHR 与周围脂质和细菌红素的特异性相互作用、膜的结构灵活性以及三聚体 NpHR 之间的相互作用可能是有效的 Cl−泵送所必需的。
We successfully reconstituted singleNatronomonas pharaonishalorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl−binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl−-releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl−uptake was faster than that of MF NpHR. These differences in the Cl−-releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl−release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl−uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl−-binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR′ state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl−pumping.