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Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids

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Title: Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids
Authors: Yamamoto, Ayumi Browse this author
Tsukamoto, Takashi Browse this author →KAKEN DB
Suzuki, Kenshiro Browse this author
Hashimoto, Eri Browse this author
Kobashigawa, Yoshihiro Browse this author
Shibasaki, Kousuke Browse this author
Uchida, Takeshi Browse this author →KAKEN DB
Inagaki, Fuyuhiko Browse this author →KAKEN DB
Demura, Makoto Browse this author →KAKEN DB
Ishimori, Koichiro Browse this author →KAKEN DB
Issue Date: 2-Jun-2020
Publisher: Cell Press
Journal Title: Biophysical journal
Volume: 118
Issue: 11
Start Page: 2853
End Page: 2865
Publisher DOI: 10.1016/j.bpj.2020.04.021
Abstract: We successfully reconstituted single Natronomonas pharaonis halorhodopsin (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 (K-d,K-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 (K-d,K-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. SIGNIFICANCE Natronomonas pharaonis halorhodopsin (NpHR), a cytoplasmic membrane Cl- pump, was reconstituted into nanodisks (NDs) using endogenous and exogenous membranes. The photoreaction cycle was examined by absorption, circular dichroism spectra, and flash photolysis. Based on the thermodynamic and kinetic parameters of the photoreaction in detergent-solubilized NpHRs, NpHRs reconstituted into artificial lipid NDs, and NpHR reconstituted into native membrane NDs, we propose that the functional differences in these NpHRs are due to 1) the low content of bacterioruberin in the membrane, 2) the different charge on the membrane surface, 3) the suppression of the conformational changes associated with the Cl- release, 4) the conformational perturbation in the NpHR trimers, and 5) the lack of the intertrimer interactions of NpHR.
Type: article
Appears in Collections:国際連携研究教育局 : GI-CoRE (Global Institution for Collaborative Research and Education : GI-CoRE) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
生命科学院・先端生命科学研究院 (Graduate School of Life Science / Faculty of Advanced Life Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

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