2004年06月, 産学連携学会, 学会賞　　　　　　　　　　　　　　　
国内学会・会議・シンポジウム等の賞, 日本国

Light-Powered Transport of Organic Anions by Microbial Rhodopsins
Simiao Shen, Shoichiro Akita, Joji Wada, Mako Eguchi, Takashi Tsukamoto, Kwang-Hwan Jung, Yuki Sudo, Takashi Kikukawa
The Journal of Physical Chemistry Letters, 16, 10528, 10535, American Chemical Society (ACS), 2025年10月02日, ［査読有り］, ［最終著者, 責任著者］
研究論文（学術雑誌）

Important amino acid residues in the chloride pump halorhodopsin that accelerate ion transport despite no direct interaction with the substrate.
Yubo Zhai, Anna Shimosaka, Takashi Tsukamoto, Takashi Kikukawa
The Journal of biological chemistry, 110703, 110703, 2025年09月11日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Ion-pump rhodopsins are widely distributed photoactive membrane proteins found in microorganisms. Their cytoplasmic (CP) regions are predominantly hydrophobic, inherently restricting substrate ion permeation. However, these rhodopsins can rapidly transport substrate ions via photo-induced conformational changes. The well-characterized H+-pumping rhodopsins employ dissociable residues such as Asp, Glu, or Lys to mediate rapid H+ relay reactions along a transiently hydrated CP pathway. In contrast, the corresponding mechanisms in other ion pumps remain poorly understood. Here, we investigated the key factors contributing to ion transport by halorhodopsin (HR), a Cl- pump from the archaeon Natronomonas pharaonis (NpHR). Upon photoactivation, NpHR creates a hydrated Cl- transport pathway in its CP region, which is surrounded by bulky hydrophobic residues that do not directly interact with Cl-. However, mutations in specific hydrophobic residues significantly slow Cl- transport. Notably, Phe211 and Leu214, located near the pathway exit, play critical roles. Mutations in these residues likely disrupt the proper positioning of the Lys215 sidechain, which inadvertently binds Cl- from the surrounding solution and positions it in a way that obstructs Cl- transport. As a result, ion passage is hindered, leading to the accumulation of long-lived intermediates. These findings suggest that the hydrophobic residues surrounding the pathway are not merely structural components. Instead, they are critical for enabling specific conformational changes that facilitate the formation of a hydrated channel, allowing efficient Cl- conduction without obstruction.

Recombinant production of isotope-labeled human α-defensin 5 via calmodulin fusion and insights into its expression enhancement.
Shaonan Yan, Hao Gu, Mitsuki Shibagaki, Jeremia Oktavian Chrisnanto, Fumi Hirai, Hiroyuki Kumeta, Yasuhiro Kumaki, Yuki Yokoi, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tatsuya Arai, Tomoyasu Aizawa
Peptides, 191, 171425, 171425, 2025年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Human α-defensin 5 (HD5), a cysteine-rich antimicrobial peptide critical for intestinal innate immunity, has been extensively studied for its structural and functional properties. Both the reduced form (HD5red) and the oxidized form (HD5oxi) exist in vivo and exhibit distinct antimicrobial activity spectra. In this study, we developed an efficient method to overexpress recombinant HD5 in Escherichia coli (E. coli) BL21 (DE3) strain by using calmodulin (CaM), which also interacts with antimicrobial peptides, as a fusion partner. Fusion expression suppressed the degradation of HD5 and reduced its toxicity to host cells. Following purification of the fusion protein and enzymatic cleavage to release the HD5 region, we successfully obtained sufficient amounts (yielding 1.5-1.7 mg/L culture) of active recombinant HD5oxi and HD5red for various applications, including stable isotope-labeled peptides for NMR analysis. Furthermore, we investigated the protective effect of CaM fusion and the mechanism of disulfide bond formation using CD and NMR spectroscopy, structural prediction, and molecular dynamics simulations. Our results suggest that the appropriate interaction strength between CaM and the HD5 region in the fusion state is a key factor for stable production.

Amphibian-Derived Cathelicidin-DM and Cathelicidin-BG: Recombinant Overexpression in Escherichia coli and Comparison of Their Structures and Antimicrobial Activities.
Chinonso Anthony Ezema, Mitsuki Shibagaki, Takashi Kikukawa, Tatsuya Arai, Tomoyasu Aizawa
ACS omega, 10, 21, 21875, 21888, 2025年06月03日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Cathelicidin-DM and cathelicidin-BG are homologous and are expected to be similar in structure and biological activity. However, while all structural prediction tools tested showed cathelicidin-BG to be helical, there were mixed results for cathelicidin-DM, with most predicting it to contain three antiparallel β-sheets and no helices. Also, separate researchers, in nonidentical conditions, reported cathelicidin-BG to possess antimicrobial activity against only Gram-positive bacteria, unlike cathelicidin-DM, which affected both Gram-positive and Gram-negative bacteria, suggesting more dissimilarities between the peptides. We therefore decided to experimentally verify and compare the structures and activities of the peptides. Until now, there is no experimentally determined structural information on either peptide, and no cheap, efficient procedure has been reported for their mass production. We hereby report a recombinant method for cathelicidin-DM and cathelicidin-BG overexpression and purification, which yielded 1.19 and 1.92 mg of pure peptides, respectively, per 0.5 L of Luria-Bertani culture. At all oxidation states, both peptides adopted a random coil structure in sodium phosphate buffer (SPB) at pH 7.4, but switched to a helical conformation in membrane mimetics. In SPB, both native peptides demonstrated strong activity against both Gram-positive and Gram-negative bacteria, with ≤5 μM of each peptide killing all cells of the tested bacterial strains, through membrane disruption as one of the possible mechanisms. We therefore conclude that, under the conditions studied, both cathelicidins have comparable structures and antimicrobial activities as their sequence homology suggested; and we recommend the use of laboratory experimentations for validation of structural prediction results.

Cryo-Raman Observation on the Photocycle of a Light-Driven Cl- Pump from Mastigocladopsis repens at High Cl- Concentration.
Kana Miyazaki, Takashi Kikukawa, Masashi Unno, Tomotsumi Fujisawa
The journal of physical chemistry. B, 129, 15, 3740, 3746, 2025年04月17日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A retinylidene photoreceptor from the cyanobacterium Mastigocladopsis repens (MrHR) is a novel type of light-driven Cl- pump that has a structural similarity to the archaeal H+ pumps but transports Cl-. An open question regarding the photocycle of this photoreceptor involves the role of a late red-shifted photoproduct, the O intermediate, which is reportedly present at high Cl- concentrations. In this study, we used cryo-Raman spectroscopy to examine the chromophore structures of the photointermediates at a high Cl- concentration. When compared to the photointermediates formed as MrHR + hv → K → L → N1 → N2 → MrHR' at a low Cl- concentration, we found that N2 kinetically disappears at a high Cl- concentration. This indicated that N2 is the transient Cl--unbound state of MrHR. In addition, we found that no Raman signal of the O intermediate is observed at the high Cl- concentration, while the signal from N1 is exclusively observed in the later stages of the photocycle. After confirming that N1's absorption spectrum displays an extending red edge, we concluded that the O intermediate is attributable to the red edge of the absorption band of N1. Here, we report a revised understanding of the photocycle for Cl- transport of MrHR.

Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy.
Xin Zhang, Hajime Tamaki, Takashi Kikukawa, Toshimichi Fujiwara, Yoh Matsuki
Biophysical chemistry, 315, 107329, 107329, 2024年12月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven Cl- inward pump that is widely used as an optogenetic tool. Although NpHR is previously extensively studied, its Cl- uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl- uptake process. In this study, we used solid-state NMR to analyze NpHR both in the Cl--bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl- depletion is widespread over the NpHR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl- uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on NpHR point mutants for the suggested residues, we confirmed their importance in the Cl- uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl--free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known NpHR crystal structures, we proposed a model that this helix movement is required for creating the Cl- entrance path on the extracellular surface of the protein and is crucial to the Cl- uptake process.

Chromophore Structural Change during the Photocycle of a Light-Driven Cl- Pump from Mastigocladopsis repens: A Cryogenic Raman Study.
Kana Miyazaki, Takashi Kikukawa, Masashi Unno, Tomotsumi Fujisawa
The journal of physical chemistry. B, 128, 40, 9692, 9698, 2024年10月10日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microbial rhodopsins are the most widely distributed photoreceptors that bind a retinal Schiff base chromophore. Among them, a light-driven Cl- pump discovered from Mastigocladopsis repens (MrHR) is distinctive in that it has the structural features of both H+ and Cl- pumps. While the photocycle has been characterized by light-induced changes of the absorption spectrum, the structural changes of the retinal chromophore remain largely unknown. In this study, we examined the chromophore structural changes of MrHR by using cryogenic Raman spectroscopy. We observed five photointermediates─K, L, N1, N2, and MrHR'─that show distinct vibrational spectra, indicating atypical chromophore structures, e.g., small distortion in the K intermediate and Schiff base configurational change in the MrHR' intermediate. Based on the Raman spectra of two N intermediates (N1 and N2), we propose that N1 is the Cl--bound state and N2 is the Cl--unbound state, which are responsible for the Cl- release and uptake, respectively, to achieve Cl- pumping.

Contribution of Proteorhodopsin to Light-Dependent Biological Responses in Hymenobacter nivis P3T Isolated from Red Snow in Antarctica.
Kaori Kondo, Ryouhei Ohtake, Shunsuke Nakano, Mia Terashima, Hisaya Kojima, Manabu Fukui, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
Biochemistry, 63, 18, 2257, 2265, 2024年09月17日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Proteorhodopsin (PR) is a major family of microbial rhodopsins that function as light-driven outward proton pumps. PR is now widely recognized for its ecological importance as a molecule responsible for solar energy flow in various ecosystems on the earth. However, few concrete examples of the actual use of light by natural microorganisms via PR have been demonstrated experimentally. This study reveals one example of that in a cryophilic bacterium Hymenobacter nivis P3T isolated from red snow in Antarctica. The results demonstrate light-dependent biochemical and biological responses in H. nivis cells, such as the proton pump activity of H. nivis PR (HnPR), which leads to the production of proton motive force, cellular ATP production, and cell growth. In addition, the results of this study demonstrate the photochemical properties of a PR, namely, HnPR, in the membrane of a natural host bacterium. The photocycle of HnPR was much faster than other PRs even at 5 °C, indicating that the proton pump function of HnPR has adapted to the low-temperature environment of Antarctica. Although it is well-known that PR helps natural host microorganisms to use light energy, this study provides another concrete example for understanding the biological role of PR by demonstrating the link between the molecular functions of PR and the light-dependent biochemical and biological responses of a PR-bearing host.

Direct detection of the chloride release and uptake reactions of Natronomonas pharaonis halorhodopsin.
Chihaya Hamada, Keisuke Murabe, Takashi Tsukamoto, Takashi Kikukawa
The Journal of biological chemistry, 300, 9, 107712, 107712, 2024年09月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Membrane transport proteins undergo multistep conformational changes to fulfill the transport of substrates across biological membranes. Substrate release and uptake are the most important events of these multistep reactions that accompany significant conformational changes. Thus, their relevant structural intermediates should be identified to better understand the molecular mechanism. However, their identifications have not been achieved for most transporters due to the difficulty of detecting the intermediates. Herein, we report the success of these identifications for a light-driven chloride transporter halorhodopsin (HR). We compared the time course of two flash-induced signals during a single transport cycle. One is a potential change of Cl--selective membrane, which enabled us to detect tiny Cl--concentration changes due to the Cl- release and the subsequent Cl--uptake reactions by HR. The other is the absorbance change of HR reflecting the sequential formations and decays of structural intermediates. Their comparison revealed not only the intermediates associated with the key reactions but also the presence of two additional Cl--binding sites on the Cl--transport pathways. The subsequent mutation studies identified one of the sites locating the protein surface on the releasing side. Thus, this determination also clarified the Cl--transport pathway from the initial binding site until the release to the medium.

A photoswitchable CENP-E inhibitor with single blue-green light to control chromosome positioning in mitotic cells.
Kazuya Matsuo, Takashi Kikukawa, Tomonori Waku, Akio Kobori, Nobuyuki Tamaoki
RSC medicinal chemistry, 2024年08月27日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Reversibly photoswitchable chemical tools have aided in the development of novel approaches in the biomedical field. The visible region of light should be ideal for the biological application of this approach because of its low phototoxicity and deep penetration depth compared to ultraviolet light. Herein, we report a photoswitchable centromere-associated protein E (CENP-E) inhibitor, which is controllable with low-energy blue-green light (around 500 nm) illumination. This photoswitchable tool enabled us to control CENP-E-driven chromosome movements and positioning at subcellular resolutions with low phototoxic effects. This study can contribute to the development of a unique technique for chromosome engineering.

Spatiotemporal regulation of CENP-E-guided chromosomes using a fast-relaxing arylazopyrazole photoswitch.
Kazuya Matsuo, Ryota Uehara, Takashi Kikukawa, Tomonori Waku, Akio Kobori, Nobuyuki Tamaoki
Chemical communications (Cambridge, England), 2024年06月07日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We developed a centromere-associated protein E (CENP-E) inhibitor employing trans to cis photoisomerization with 405 nm visible light illumination and fast thermal relaxation. This photoswitching characteristic of the inhibitor enabled selective blockage or release of the motion of particular chromosomes within a single mitotic cell. Using this technique, we successfully demonstrated targeted chromosome gain and loss in daughter cells by introducing asymmetric chromosome segregation.

Configurational Changes of Retinal Schiff Base during Membrane Na+ Transport by a Sodium Pumping Rhodopsin.
Tomotsumi Fujisawa, Kouta Kinoue, Ryouhei Seike, Takashi Kikukawa, Masashi Unno
The journal of physical chemistry letters, 15, 7, 1993, 1998, 2024年02月22日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microbial rhodopsins are photoreceptors containing the retinal Schiff base chromophore and are ubiquitous among microorganisms. The Schiff base configuration of the chromophore, 15-anti (C═N trans) or 15-syn (C═N cis), is structurally important for their functions, such as membrane ion transport, because this configuration dictates the orientation of the positively charged NH group that interacts with substrate ions. The 15-anti/syn configuration is thus essential for elucidating the ion-transport mechanisms in microbial rhodopsins. Here, we identified the Schiff base configuration during the photoreaction of a sodium pumping rhodopsin from Indibacter alkaliphilus using Raman spectroscopy. We found that the unique configurational change from the 13-cis, 15-anti to all-trans, 15-syn form occurs between the photointermediates termed O1 and O2, which accomplish the Na+ uptake and release, respectively. This isomerization is considered to give rise to the highly irreversible O1 → O2 step that is crucial for unidirectional Na+ transport.

Multistep conformational changes leading to the gate opening of light-driven sodium pump rhodopsin.
Yukino Sato, Tsubasa Hashimoto, Koji Kato, Akiko Okamura, Kaito Hasegawa, Tsukasa Shinone, Yoshikazu Tanaka, Yoshiki Tanaka, Tomoya Tsukazaki, Takashi Tsukamoto, Makoto Demura, Min Yao, Takashi Kikukawa
The Journal of biological chemistry, 105393, 105393, 2023年10月25日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Membrane transport proteins require a gating mechanism that opens and closes the substrate transport pathway to carry out unidirectional transport. The "gating" involves large conformational changes and is achieved via multistep reactions. However, these elementary steps have not been clarified for most transporters due to the difficulty of detecting the individual steps. Here, we propose these steps for the gate opening of the bacterial Na+ pump rhodopsin (NaR), which outwardly pumps Na+ upon illumination. We herein solved an asymmetric dimer structure of NaR from the bacterium Indibacter alkaliphilus. In one protomer, the Arg108 sidechain is oriented toward the protein center and appears to block a Na+ release pathway to the extracellular (EC) medium. In the other protomer, however, this sidechain swings to the EC side and then opens the release pathway. Assuming that the latter protomer mimics the Na+-releasing intermediate, we examined the mechanism for the swing motion of the Arg108 sidechain. On the EC surface of the first protomer, there is a characteristic cluster consisting of Glu10, Glu159, and Arg242 residues connecting three helices. In contrast, this cluster is disrupted in the second protomer. Our experimental results suggested that this disruption is a key process. The cluster disruption induces the outward movement of the Glu159-Arg242 pair and simultaneously rotates the seventh transmembrane helix. This rotation resultantly opens a space for the swing motion of the Arg108 sidechain. Thus, cluster disruption might occur during the photoreaction and then trigger sequential conformation changes leading to the gate-open state.

The preferential transport of NO3- by full-length Guillardia theta anion channelrhodopsin 1 is enhanced by its extended cytoplasmic domain.
Yuya Ohki, Tsukasa Shinone, Sayo Inoko, Miu Sudo, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
The Journal of biological chemistry, 105305, 105305, 2023年09月29日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Previous research of anion channelrhodopsins (ACRs) has been performed using cytoplasmic domain (CPD)-deleted constructs, and therefore have overlooked the native functions of full-length ACRs and the potential functional role(s) of the CPD. In this study, we used the recombinant expression of full-length Guillardia theta ACR1 (GtACR1_full) for pH measurements in Pichia pastoris cell suspensions as an indirect method to assess its anion transport activity, and for absorption spectroscopy and flash photolysis characterization of the purified protein. The results show that the CPD, which was predicted to be intrinsically disordered and possibly phosphorylated, enhanced NO3- transport compared to Cl- transport, which resulted in the preferential transport of NO3-. This correlated with the extended lifetime and large accumulation of the photocycle intermediate that is involved in the gate-open state. Considering that the depletion of a nitrogen source enhances the expression of GtACR1 in native algal cells, we suggest that NO3- transport could be the natural function of GtACR1_full in algal cells.

A Basic Study of the Effects of Mulberry Leaf Administration to Healthy C57BL/6 Mice on Gut Microbiota and Metabolites
Li Gan, Yuga Inamura, Yu Shimizu, Yuki Yokoi, Yuki Ohnishi, Zihao Song, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Masaaki Ito, Tokiyoshi Ayabe, Kiminori Nakamura, Tomoyasu Aizawa
Metabolites, 13, 9, 1003, 1003, MDPI AG, 2023年09月10日, ［査読有り］
研究論文（学術雑誌）, Mulberry leaves contain α-glucosidase inhibitors, which have hypoglycemic effects and are considered functional foods. However, few reports have covered the effects of mulberry leaf components on normal gut microbiota and gut metabolites. Herein, gut microbiota analysis and NMR-based metabolomics were performed on the feces of mulberry leaf powder (MLP)-treated mice to determine the effects of long-term MLP consumption. Gut microbiota in the mouse were analyzed using 16S-rRNA gene sequencing, and no significant differences were revealed in the diversity and community structure of the gut microbiota in the C57BL/6 mice with or without MLP supplementation. Thirty-nine metabolites were identified via 1H-NMR analysis, and carbohydrates and amino acids were significantly (p < 0.01–0.05) altered upon MLP treatment. In the MLP-treated group, there was a marked increase and decrease in maltose and glucose concentrations, respectively, possibly due to the degradation inhibitory activity of oligosaccharides. After 5 weeks, all amino acid concentrations decreased. Furthermore, despite clear fluctuations in fecal saccharide concentrations, short-chain fatty acid production via intestinal bacterial metabolism was not strongly affected. This study provides the knowledge that MLP administration can alter the gut metabolites without affecting the normal gut microbiota, which is useful for considering MLP as a healthy food source.

Key determinants for signaling in the sensory rhodopsin II/transducer complex are different between Halobacterium salinarum and Natronomonas pharaonis.
Risa Matsunami-Nakamura, Jun Tamogami, Miki Takeguchi, Junya Ishikawa, Takashi Kikukawa, Naoki Kamo, Toshifumi Nara
FEBS letters, 2023年08月02日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The cell membrane of Halobacterium salinarum contains a retinal-binding photoreceptor, sensory rhodopsin II (HsSRII), coupled with its cognate transducer (HsHtrII), allowing repellent phototaxis behavior for shorter wavelength light. Previous studies on SRII from Natronomonas pharaonis (NpSRII) pointed out the importance of the hydrogen bonding interaction between Thr204NpSRII and Tyr174NpSRII in signal transfer from SRII to HtrII. Here, we investigated the effect on phototactic function by replacing residues in HsSRII corresponding to Thr204NpSRII and Tyr174NpSRII . Whereas replacement of either residue altered the photocycle kinetics, introduction of any mutations at Ser201HsSRII and Tyr171HsSRII did not eliminate negative phototaxis function. These observations imply the possibility of the presence of an unidentified molecular mechanism for photophobic signal transduction differing from NpSRII-NpHtrII.

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions
Yi Wang, Yuchi Song, Shaonan Yan, Rina Hiramine, Yuki Ohnishi, Yuki Yokoi, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa
Antibiotics, 12, 6, 1047, 1047, MDPI AG, 2023年06月14日, ［査読有り］
研究論文（学術雑誌）, Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.

Elucidation of a Thermodynamical Feature Attributed to Substrate Binding to the Prokaryotic H+/Oligopeptide Cotransporter YdgR with Calorimetric Analysis: The Substrate Binding Driven by the Change in Entropy Implies the Release of Bound Water Molecules from the Binding Pocket.
Akiko Omori, Shotaro Sasaki, Takashi Kikukawa, Kazumi Shimono, Seiji Miyauchi
Biochemistry, 62, 11, 1608, 1618, 2023年06月06日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Here, we have elucidated the substrate recognition mechanism by a prokaryotic H+/oligopeptide cotransporter, YdgR, using isothermal titration calorimetry. Under acidic conditions (pH 6.0), the binding of a dipeptide, Val-Ala, to YdgR elicited endothermic enthalpy, which compensated for the increase in entropy due to dipeptide binding. A series of dipeptides were used in the binding titration. The dipeptides represent Val-X and X-Val, where X is Ala, Ser, Val, Tyr, or Phe. Most dipeptides revealed endothermic enthalpy, which was completely compensated by the increase in entropy due to dipeptide binding. The change in enthalpy due to binding correlated well with the change in entropy, whereas the Gibbs free energy involved in the binding of the dipeptide to YdgR remained unchanged irrespective of dipeptide sequences, implying that the binding reaction was driven by entropy, that is, the release of bound water molecules in the binding pocket. It is also important to clarify that, based on the prediction of water molecules in the ligand-binding pocket of YdgR, the release of three bound water molecules in the putative substrate binding pocket occurred through binding to YdgR. In the comparison of Val-X and X-Val dipeptides, the N-terminal region of the binding pocket might contain more bound water molecules than the C-terminal region. In light of these findings, we suggest that bound water molecules might play an important role in substrate recognition and binding by YdgR.

Structure and Heterogeneity of Retinal Chromophore in Chloride Pump Rhodopsins Revealed by Raman Optical Activity.
Masaiku Ohya, Takashi Kikukawa, Junpei Matsuo, Takashi Tsukamoto, Ryota Nagaura, Tomotsumi Fujisawa, Masashi Unno
The journal of physical chemistry. B, 2023年05月18日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Chloride transport by microbial rhodopsins is actively being researched to understand how light energy is converted to drive ion pumping across cell membranes. Chloride pumps have been identified in archaea and eubacteria, and there are similarities and differences in the active site structures between these groups. Thus, it has not been clarified whether a common mechanism underlies the ion pump processes for all chloride-pumping rhodopsins. Here, we applied Raman optical activity (ROA) spectroscopy to two chloride pumps, Nonlabens marinus rhodopsin-3 (NM-R3) and halorhodopsin from the cyanobacterium Mastigocladopsis repens (MrHR). ROA is a vibrational spectroscopy that provides chiral sensitivity, and the sign of ROA signals can reveal twisting of cofactor molecules within proteins. Our ROA analysis revealed that the retinal Schiff base NH group orients toward the C helix and forms a direct hydrogen bond with a nearby chloride ion in NM-R3. In contrast, MrHR is suggested to contain two retinal conformations twisted in opposite directions; one conformation has a hydrogen bond with a chloride ion like NM-R3, while the other forms a hydrogen bond with a water molecule anchored by a G helix residue. These results suggest a general pump mechanism in which the chloride ion is "dragged" by the flipping Schiff base NH group upon photoisomerization.

Phenylazothiazoles as Visible-Light Photoswitches
Runze Lin, P. K. Hashim, Saugata Sahu, Ammathnadu S. Amrutha, Nusaiba Madappuram Cheruthu, Shakkeeb Thazhathethil, Kiyonori Takahashi, Takayoshi Nakamura, Takashi Kikukawa, Nobuyuki Tamaoki
Journal of the American Chemical Society, American Chemical Society (ACS), 2023年04月12日, ［査読有り］
研究論文（学術雑誌）

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.
Yuchi Song, Yi Wang, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa
Microbial cell factories, 22, 1, 9, 9, 2023年01月13日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

Mutations conferring SO42- pumping ability on the cyanobacterial anion pump rhodopsin and the resultant unique features of the mutant.
Yuhei Doi, Jo Watanabe, Ryota Nii, Takashi Tsukamoto, Makoto Demura, Yuki Sudo, Takashi Kikukawa
Scientific reports, 12, 1, 16422, 16422, 2022年09月30日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Membrane transport proteins can be divided into two types: those that bind substrates in a resting state and those that do not. In this study, we demonstrate that these types can be converted by mutations through a study of two cyanobacterial anion-pumping rhodopsins, Mastigocladopsis repens halorhodopsin (MrHR) and Synechocystis halorhodopsin (SyHR). Anion pump rhodopsins, including MrHR and SyHR, initially bind substrate anions to the protein center and transport them upon illumination. MrHR transports only smaller halide ions, Cl- and Br-, but SyHR also transports SO42-, despite the close sequence similarity to MrHR. We sought a determinant that could confer SO42- pumping ability on MrHR and found that the removal of a negative charge at the anion entrance is a prerequisite for SO42- transport by MrHR. Consistently, the reverse mutation in SyHR significantly weakened SO42- pump activity. Notably, the MrHR and SyHR mutants did not show SO42- induced absorption spectral shifts or changes in the photoreactions, suggesting no bindings of SO42- in their initial states or the bindings to the sites far from the protein centers. In other words, unlike wild-type SyHR, these mutants take up SO42- into their centers after illumination and release it before the ends of the photoreactions.

Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues
Hao Gu, Takasumi Kato, Hiroyuki Kumeta, Yasuhiro Kumaki, Takashi Tsukamoto, Takashi Kikukawa, Makoto Demura, Hiroaki Ishida, Hans J. Vogel, Tomoyasu Aizawa
ACS Omega, 7, 36, 31924, 31934, American Chemical Society (ACS), 2022年09月02日, ［査読有り］
研究論文（学術雑誌）

Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin.
Tomotsumi Fujisawa, Kouta Kinoue, Ryouhei Seike, Takashi Kikukawa, Masashi Unno
The Journal of biological chemistry, 298, 9, 102366, 102366, 2022年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Sodium-pumping rhodopsins (NaRs) are membrane transporters that utilize light energy to pump Na+ across the cellular membrane. Within the NaRs, the retinal Schiff base chromophore absorbs light, and a photochemically induced transient state, referred to as the "O intermediate", performs both the uptake and release of Na+. However, the structure of the O intermediate remains unclear. Here, we used time-resolved cryo-Raman spectroscopy under preresonance conditions to study the structure of the retinal chromophore in the O intermediate of an NaR from the bacterium Indibacter alkaliphilus. We observed two O intermediates, termed O1 and O2, having distinct chromophore structures. We show O1 displays a distorted 13-cis chromophore, while O2 contains a distorted all-trans structure. This finding indicated that the uptake and release of Na+ are achieved not by a single O intermediate but by two sequential O intermediates that are toggled via isomerization of the retinal chromophore. These results provide crucial structural insight into the unidirectional Na+ transport mediated by the chromophore-binding pocket of NaRs.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.
Yuji Sato, Yi Wang, Yuchi Song, Weiming Geng, Shaonan Yan, Kiminori Nakamura, Takashi Kikukawa, Makoto Demura, Tokiyoshi Ayabe, Tomoyasu Aizawa
Amino acids, 54, 289, 297, 2022年01月17日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Pore Size Distributions Related with Spontaneous Purple Membrane Stacking in Porous Hydrogels
Morise Karasawa, Yasunori Yokoyama, Kingo Takiguchi, Hiroshi Takahashi, Takashi Kikukawa, Masashi Sonoyama, Koshi Takenaka
Journal of the Physical Society of Japan, 90, 10, 103801, 103801, Physical Society of Japan, 2021年10月15日, ［査読有り］, ［国内誌］
研究論文（学術雑誌）

Comparison of functionality and structural stability of bacteriorhodopsin reconstituted in partially fluorinated dimyristoylphosphatidylcholine liposomes with different perfluoroalkyl chain lengths.
Mami Hashimoto, Yuka Murai, Kohei Morita, Takashi Kikukawa, Toshiyuki Takagi, Hiroshi Takahashi, Yasunori Yokoyama, Hideki Amii, Masashi Sonoyama
Biochimica et biophysica acta. Biomembranes, 1863, 10, 183686, 183686, 2021年10月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Amphiphilic molecules with one or more perfluoroalkyl groups (Rf, CnF2n+1), which show peculiar interfacial properties, are attracting much attention in membrane protein science. We recently have developed a partially fluorinated dimyristoylphosphatidylcholine (DMPC) with a perfluorobutyl group in the hydrophobic chain terminal (F4-DMPC) and demonstrated that F4-DMPC is a promising material for incorporating membrane proteins. Moreover, we have found out that membrane properties of a series of partially fluorinated DMPCs with different Rf chain lengths (Fn-DMPCs) vary in a significant Rf chain length-dependent manner. In the present study, structural and functional properties of a membrane protein bacteriorhodopsin (bR) in the Fn-DMPC (n = 4, 6, and 8) membranes (bR/Fn-DMPC) are investigated using several physicochemical techniques. Regardless of the Rf chain lengths, bR/Fn-DMPCs retain native-like structural and functional properties at 30 °C, unlike bR molecules in DMPC vesicles. In particular, bR/F6-DMPC, which is in the fluid phase at 30 °C, shows flash-induced transient absorption changes very similar to the native purple membrane (PM) and very high thermal stability of bR trimers comparable to the PM. Structural and functional properties of bR/Fn-DMPCs are discussed compared to the PM and bR/DMPC.

Replaceability of Schiff base proton donors in light-driven proton pump rhodopsins.
Syogo Sasaki, Jun Tamogami, Koki Nishiya, Makoto Demura, Takashi Kikukawa
The Journal of biological chemistry, 297, 3, 101013, 101013, 2021年07月28日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Many H+-pump rhodopsins conserve "H+ donor" residues in cytoplasmic (CP) half channels to quickly transport H+ from the CP medium to Schiff bases at the center of these proteins. For conventional H+ pumps, the donors are conserved as Asp or Glu but are replaced by Lys in the minority, such as Exiguobacterium sibiricum rhodopsin (ESR). In dark states, carboxyl donors are protonated, whereas the Lys donor is deprotonated. As a result, carboxyl donors first donate H+ to the Schiff bases and then capture the other H+ from the medium, whereas the Lys donor first captures H+ from the medium and then donates it to the Schiff base. Thus, carboxyl and Lys-type H+ pumps seem to have different mechanisms, which are probably optimized for their respective H+-transfer reactions. Here, we examined these differences via replacement of donor residues. For Asp-type deltarhodopsin (DR), the embedded Lys residue distorted the protein conformation and did not act as the H+ donor. In contrast, for Glu-type proteorhodopsin (PR) and ESR, the embedded residues functioned well as H+ donors. These differences were further examined by focusing on the activation volumes during the H+-transfer reactions. The results revealed essential differences between archaeal H+ pump (DR) and eubacterial H+ pumps PR and ESR. Archaeal DR requires significant hydration of the CP channel for the H+-transfer reactions; however, eubacterial PR and ESR require the swing-like motion of the donor residue rather than hydration. Given this common mechanism, donor residues might be replaceable between eubacterial PR and ESR.

Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization.
Masuzu Kikuchi, Keiichi Kojima, Shin Nakao, Susumu Yoshizawa, Shiho Kawanishi, Atsushi Shibukawa, Takashi Kikukawa, Yuki Sudo
Scientific reports, 11, 1, 14765, 14765, 2021年07月20日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Preference of Proteomonas sulcata anion channelrhodopsin for NO3- revealed using a pH electrode method.
Chihiro Kikuchi, Hina Kurane, Takuma Watanabe, Makoto Demura, Takashi Kikukawa, Takashi Tsukamoto
Scientific reports, 11, 1, 7908, 7908, 2021年04月12日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Ion channel proteins are physiologically important molecules in living organisms. Their molecular functions have been investigated using electrophysiological methods, which enable quantitative, precise and advanced measurements and thus require complex instruments and experienced operators. For simpler and easier measurements, we measured the anion transport activity of light-gated anion channelrhodopsins (ACRs) using a pH electrode method, which has already been established for ion pump rhodopsins. Using that method, we successfully measured the anion transport activity and its dependence on the wavelength of light, i.e. its action spectra, and on the anion species, i.e. its selectivity or preference, of several ACRs expressed in yeast cells. In addition, we identified the strong anion transport activity and the preference for NO3- of an ACR from a marine cryptophyte algae Proteomonas sulcata, named PsuACR_353. Such a preference was discovered for the first time in microbial pump- or channel-type rhodopsins. Nitrate is one of the most stable forms of nitrogen and is used as a nitrogen source by most organisms including plants. Therefore, PsuACR_353 may play a role in NO3- transport and might take part in NO3--related cellular functions in nature. Measurements of a mutant protein revealed that a Thr residue in the 3rd transmembrane helix, which corresponds to Cys102 in GtACR1, contributed to the preference for NO3-. These findings will be helpful to understand the mechanisms of anion transport, selectivity and preference of PsuACR_353.

Low-temperature Raman spectroscopy of sodium-pump rhodopsin from Indibacter alkaliphilus: insight of Na+ binding for active Na+ transport.
Yushi Nakamizo, Tomotsumi Fujisawa, Takashi Kikukawa, Akiko Okamura, Hiroaki Baba, Masashi Unno
Physical chemistry chemical physics : PCCP, 23, 3, 2072, 2079, 2021年01月28日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We carried out the low-temperature Raman measurement of a sodium pump rhodopsin from Indibacter alkaliphilus (IaNaR) and examined the primary structural change for the light-driven Na+ pump. We observed that photoexcitation of IaNaR produced the distorted 13-cis retinal chromophore in the presence of Na+, while the structural distortion was significantly relaxed in the absence of Na+. This structural difference of the chromophore with/without Na+ was attributed to the Na+ binding to the protein, which alters the active site. Using the spectral sensitivity to the ion binding, we found that IaNaR had a second Na+ binding site in addition to the one already specified on the extracellular surface. To date, the Na+ binding has not been considered as a prerequisite for Na+ transport. However, this study provides insight that the protein structural change induced by the ion binding involved the formation of an R108-D250 salt bridge, which has critical importance in the active transport of Na+.

Cl^–ポンプロドプシンの分子機構とH⁺ポンプへの機能変換
菊川 峰志
生物物理, 61, 1, 012, 015, 一般社団法人 日本生物物理学会, 2021年, ［査読有り］, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
日本語, Cl^–-pump rhodopsin is the second discovered microbial rhodopsin and functions as light-driven Cl^– pump. The physiological significance of the Cl^– pump has not been fully resolved. However, its functional mechanism has been studied as a model system of anion transporters. In this review, the variation and functional mechanisms of Cl^–-pump rhodopsins were summarized. After 2014, novel Cl^–-pump groups were discovered in marine and terrestrial bacteria and were revealed to have unique characteristics. The most recently identified protein has close similarity with the H⁺-pump rhodopsin and begins to pump H⁺ outwardly by only a single amino acid replacement.

Unique Cl^– pump rhodopsin with close similarity to H⁺ pump rhodopsin
Takashi Kikukawa
Biophysics and Physicobiology, 18, 317, 326, Biophysical Society of Japan, 2021年, ［査読有り］, ［招待有り］, ［筆頭著者, 最終著者, 責任著者］
英語, 研究論文（学術雑誌）

Spectroscopic approach for exploring structure and function of photoreceptor proteins.
Masashi Unno, Yuu Hirose, Masaki Mishima, Takashi Kikukawa, Tomotsumi Fujisawa, Tatsuya Iwata, Jun Tamogami
Biophysics and physicobiology, 18, 127, 130, 2021年, ［査読有り］, ［招待有り］, ［国内誌］
英語, 研究論文（学術雑誌）

Protonation State of a Histidine Residue in Human Oligopeptide Transporter 1 (hPEPT1) Regulates hPEPT1-Mediated Efflux Activity.
Akiko Omori, Yuki Fujisawa, Shotaro Sasaki, Kazumi Shimono, Takashi Kikukawa, Seiji Miyauchi
Biological & pharmaceutical bulletin, 44, 5, 678, 685, 2021年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, To clarify the role of an amino acid residue in the pH-dependent efflux process in Chinese hamster ovary (CHO) cells expressing the human oligopeptide transporter hPEPT1 (CHO/hPEPT1), we determined the effect of extracellular pH on the hPEPT1-mediated efflux process. The efflux of glycylsarcosine (Gly-Sar), a typical substrate for hPEPT1, was determined using an infinite dilution method after cells were preloaded with [3H]-Gly-Sar. The efflux of [3H]-Gly-Sar was stimulated by 5 mM unlabeled hPEPT1 substrates in the medium. This trans-stimulation phenomenon showed that hPEPT1 mediated the efflux of [3H]-Gly-Sar from CHO/hPEPT1 and that hPEPT1 is a bi-directional transporter. We then determined the effect of extracellular pH (varying from 8.0 to 3.5) on the efflux activity. The efflux activity by hPEPT1 decreased with the decrease in extracellular pH. The Henderson-Hasselbälch-type equation, which fitted well to the pH-profile of efflux activity, indicated that a single amino acid residue with a pKa value of approximately 5.7 regulates the efflux activity. The pH-profile of the efflux activity remained almost unchanged irrespective of the proton gradient across the plasma membrane. In addition, the chemical modification of the histidine residue with diethylpyrocarbonate completely abolished the efflux activity from cells, which could be prevented by the presence of 10 mM Gly-Sar. These data indicate that the efflux process of hPEPT1 is also regulated in a pH-dependent manner by the protonation state of a histidine residue located at or near the substrate recognition site facing the extracellular space.

Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin
Tomoya Kato, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa
Journal of Biological Chemistry, 296, 100792, 100792, Elsevier BV, 2021年01月, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Membrane transport proteins undergo critical conformational changes during substrate uptake and release, as the substrate-binding site is believed to switch its accessibility from one side of the membrane to the other. Thus, at least two substrate-binding intermediates should appear during the process, that is, after uptake and before the release of the substrate. However, this view has not been verified for most transporters due to the difficulty in detecting short-lived intermediates. Here, we report real-time identification of these intermediates for the light-driven outward current-generating Na+ pump rhodopsin (NaR). We triggered the transport cycle of NaR using a short laser pulse, and subsequent formation and decay of various intermediates was detected by time-resolved measurements of absorption changes. We used this method to analyze transport reactions, and elucidated the sequential formation of the Na+-binding intermediates O1 and O2. Both intermediates exhibited red-shifted absorption spectra and generated transient equilibria with short-wavelength intermediates. The equilibria commonly shifted toward O1 and O2 with increasing Na+ concentration, indicating that Na+ is bound to these intermediates. However, these equilibria were formed independently; O1 reached equilibrium with preceding intermediates, indicating Na+ uptake on the cytoplasmic side. In contrast, O2 reached equilibrium with subsequent intermediates, indicating Na+ release on the extracellular side. Thus, there is an irreversible switch in "accessibility" during the O1 to O2 transition, which could represent one of the key processes governing unidirectional Na+ transport.

Effects of salt and gel network structures on purple membrane stacking in hydrogels immobilized with poly(vinyl alcohol)
Yasunori Yokoyama, Shunsuke Yano, Riku Kurita, Morise Karasawa, Hikaru Tanaka, Hiroshi Takahashi, Takashi Kikukawa, Masashi Sonoyama, Koshi Takenaka
JOURNAL OF APPLIED PHYSICS, 129, 1, 2021年01月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift.
Keiichi Kojima, Susumu Yoshizawa, Masumi Hasegawa, Masaki Nakama, Marie Kurihara, Takashi Kikukawa, Yuki Sudo
Scientific reports, 10, 1, 20857, 20857, 2020年11月30日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named 'schizorhodopsin (SzR)' has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pKa = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pKa = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral 'blue-shift' due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

"Watching" a Molecular Twist in a Protein by Raman Optical Activity.
Junpei Matsuo, Takashi Kikukawa, Tomotsumi Fujisawa, Wouter D Hoff, Masashi Unno
The journal of physical chemistry letters, 11, 20, 8579, 8584, 2020年09月25日, ［査読有り］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）, Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.

Direct Detection of the Substrate Uptake and Release Reactions of the Light-Driven Sodium-Pump Rhodopsin.
Keisuke Murabe, Takashi Tsukamoto, Tomoyasu Aizawa, Makoto Demura, Takashi Kikukawa
Journal of the American Chemical Society, 142, 37, 16023, 16030, 2020年09月16日, ［査読有り］, ［最終著者, 責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, For membrane transporters, substrate uptake and release reactions are major events during their transport cycles. Despite the functional importance of these events, it is difficult to identify their relevant structural intermediates because of the requirements of the experimental methods, which are to detect the timing of the formation and decay of intermediates and to detect the timing of substrate uptake and release. We report successfully achieving this for the light-driven Na+ pump rhodopsin (NaR). Here, a Na+-selective membrane, which consists of polyvinyl chloride and a Na+ ionophore, was employed to detect Na+ uptake and release. When one side of the membrane was covered by the lipid-reconstituted NaR, continuous illumination induced an increase in membrane potential, which reflected Na+ uptake by the photolyzed NaR. Via use of nanosecond laser pulses, two kinds of data were obtained during a single transport cycle: one was the flash-induced absorbance change in NaR to detect the formation and decay of structural intermediates, and the other was the flash-induced change in membrane potential, which reflects the transient Na+ uptake and release reactions. Their comparison clearly indicated that Na+ is captured and released during the formation and decay of the O intermediate, the red-shifted intermediate that appears in the latter half of the transport cycle.

Photoisomerization of azobenzene units drives the photochemical reaction cycles of proteorhodopsin and bacteriorhodopsin analogues.
Shariful Haque, Takashi Kikukawa, Nobuyuki Tamaoki
Organic & biomolecular chemistry, 18, 32, 6312, 6327, 2020年08月19日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, In this study we substituted the retinal units in proteorhodopsin (PR) and bacteriorhodopsin (BR) with azo chromophores to investigate the mechanism of photoinduced proton pumping in rhodopsins and potentially develop new artificial molecular pumps. We used an indium tin oxide electrode to investigate the photoinduced proton transfer of the azo analogues of PR and BR. We also employed flash photolysis to determine the characteristic photocycles, comprising multiple transient intermediates, of the azo chromophore-bound PR and BR. Moreover, our studies of the photoinduced proton pumping functions of azo-proteoopsin and azo-bacterioopsin complexes revealed that they did not pump protons upon illumination, even though they underwent photoinduced proton transfer and the characteristic photocycle. Mutational analysis suggested that the proton pumping malfunction of the azo analogues of PR and BR resulted from the absence of proton transfer reactions through cytoplasmic channels, even though these reactions were evoked in extracellular channels. Based on our experimental findings, we propose herein a putative model of the proton transfer reaction mechanism for the azo analogues of PR and BR.

Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome.
Sato T, Kikukawa T, Miyoshi R, Kajimoto K, Yonekawa C, Fujisawa T, Unno M, Eki T, Hirose Y
The Journal of biological chemistry, 294, 49, 18909, 18922, 2019年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state (
15Z
Pb) and orange-absorbing 15E state (
15E
Po). pH titration analysis revealed that
15Z
Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy,
15E
Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state (
15Z
Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in
15Z
Pb and bilin protonation in
15E
Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated
15Z
Pg and protonated
15E
Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Time-resolved spectroscopy revealed
15Z
Pg formation as an intermediate in the
15E
Pr-to-
15Z
Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blue-absorbing CBCR subfamilies.

Low-Temperature Raman Spectroscopy of Halorhodopsin from Natronomonas pharaonis: Structural Discrimination of Blue-Shifted and Red-Shifted Photoproducts.
Fujisawa T, Kiyota H, Kikukawa T, Unno M
Biochemistry, 58, 40, 4159, 4167, 2019年10月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Functional importance of the oligomer formation of the cyanobacterial H⁺ pump Gloeobacter rhodopsin.
Iizuka A, Kajimoto K, Fujisawa T, Tsukamoto T, Aizawa T, Kamo N, Jung KH, Unno M, Demura M, Kikukawa T
Scientific reports, 9, 1, 10711, 2019年07月, ［査読有り］, ［最終著者, 責任著者］, ［国際共著］, ［国際誌］
英語, 研究論文（学術雑誌）

Comparison of two different partially fluorinated phosphatidylcholines with the perfluorobutyl group on thermotropic properties of the bilayer membrane and reconstituted bacteriorhodopsin.
Yanagi T, Takagi T, Takahashi H, Kikukawa T, Amii H, Sonoyama M
Biophysical reviews, 11, 3, 395, 398, Springer Science and Business Media LLC, 2019年05月, ［査読有り］, ［国際誌］
研究論文（学術雑誌）

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers.
Takahashi H, Yoshino M, Morita K, Takagi T, Yokoyama Y, Kikukawa T, Amii H, Kanamori T, Sonoyama M
Biochimica et biophysica acta. Biomembranes, 1861, 3, 631, 642, 2019年03月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin.
Hasemi T, Kikukawa T, Watanabe Y, Aizawa T, Miyauchi S, Kamo N, Demura M
Biochimica et biophysica acta. Bioenergetics, 1860, 2, 136, 146, 2019年02月, ［査読有り］, ［責任著者］, ［国際誌］
英語, 研究論文（学術雑誌）, Mastigocladopsis repens halorhodopsin (MrHR) is a Cl--pumping rhodopsin that belongs to a distinct cluster far from other Cl- pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I- similar to Cl- but cannot transport it. I--bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl--pumping photocycle, Cl- moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I--bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl--binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl--binding affinity nor the absorption spectrum, but completely eliminated the Cl--pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H+ release was detected for photolyzed MrHR by using an indium‑tin oxide electrode, which acts as a good time-resolved pH sensor. This H+ release disappeared in the I--bound form of the wild-type and Cl--bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl- movement to the CP channel.

Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle.
Geng X, Dai G, Chao L, Wen D, Kikukawa T, Iwasa T
Photochemistry and photobiology, 95, 4, 980, 989, 2018年12月, ［査読有り］, ［国際共著］, ［国際誌］

Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl^- concentrations.
Tsukamoto T, Kikuchi C, Suzuki H, Aizawa T, Kikukawa T, Demura M
Scientific reports, 8, 1, 13445, 13445, 2018年09月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl- dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl- in the initial state with a Kd of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl- concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl- non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl- conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl-.

Low-temperature Raman spectroscopy reveals small chromophore distortion in primary photointermediate of proteorhodopsin.
Fujisawa T, Abe M, Tamogami J, Kikukawa T, Kamo N, Unno M
FEBS letters, 592, 18, 3054, 3061, 2018年08月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Interhelical interactions between D92 and C218 in the cytoplasmic domain regulate proton uptake upon N-decay in the proton transport of Acetabularia rhodopsin II
Jun Tamogami, Takashi Kikukawa, Keisuke Ohkawa, Noboru Ohsawa, Toshifumi Nara, Makoto Demura, Seiji Miyauchi, Tomomi Kimura-Someya, Mikako Shirouzu, Shigeyuki Yokoyama, Kazumi Shimono, Naoki Kamo
Journal of Photochemistry and Photobiology B: Biology, 183, 35, 45, Elsevier B.V., 2018年06月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）

Presence of a Haloarchaeal Halorhodopsin-Like Cl- Pump in Marine Bacteria.
Yu Nakajima, Takashi Tsukamoto, Yohei Kumagai, Yoshitoshi Ogura, Tetsuya Hayashi, Jaeho Song, Takashi Kikukawa, Makoto Demura, Kazuhiro Kogure, Yuki Sudo, Susumu Yoshizawa
Microbes and environments, 33, 1, 89, 97, 2018年03月29日, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl--pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl- pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl--transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl--pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.

Photocycle of Sensory Rhodopsin II from Halobacterium salinarum (HsSRII): Mutation of D103 Accelerates M Decay and Changes the Decay Pathway of a 13-cis O-like Species.
Dai G, Geng X, Chao L, Tamogami J, Kikukawa T, Demura M, Kamo N, Iwasa T
Photochemistry and photobiology, 94, 4, 705, 714, 2018年03月, ［査読有り］, ［国際共著］, ［国際誌］

Spectroscopic characteristics of Rubricoccus marinus xenorhodopsin (RmXeR) and a putative model for its inward H+ transport mechanism.
Saki Inoue, Susumu Yoshizawa, Yu Nakajima, Keiichi Kojima, Takashi Tsukamoto, Takashi Kikukawa, Yuki Sudo
Physical chemistry chemical physics : PCCP, 20, 5, 3172, 3183, 2018年01月31日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, A new group of microbial rhodopsins named xenorhodopsins (XeR), which are closely related to the cyanobacterial Anabaena sensory rhodopsin, show a light-driven "inward" proton transport activity, as reported for one representative of this group from Parvularcula oceani (PoXeR). In this study, we functionally and spectroscopically characterized a new member of the XeR clade from a marine bacterium Rubricoccus marinus SG-29T (RmXeR). Escherichia coli cells expressing recombinant RmXeR showed a light-induced alkalization of the cell suspension, which was strongly impaired by a protonophore, suggesting that RmXeR is a light-driven "inward" proton pump as is PoXeR. The spectroscopic properties of purified RmXeR were investigated and compared with those of PoXeR and a light-driven "outward" proton pump, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum. Action spectroscopy revealed that RmXeR with all-trans retinal is responsible for the light-driven inward proton transport activity, but not with 13-cis retinal. From pH titration experiments and mutational analysis, we estimated the pKa values for the protonated Schiff base of the retinal chromophore and its counterion as 11.1 ± 0.07 and 2.1 ± 0.07, respectively. Of note, the direction of both the retinal composition change upon light-dark adaptation and the acid-induced spectral shift was opposite that of BR, which is presumably related to the opposite directions of ion transport (from outside to inside for RmXeR and from inside to outside for BR). Flash photolysis experiments revealed the appearances of three intermediates (L, M and O) during the photocycle. The proton uptake and release were coincident with the formation and decay of the M intermediate, respectively. Together with associated findings from other microbial rhodopsins, we propose a putative model for the inward proton transport mechanism of RmXeR.

Targeted Activation of Molecular Transportation by Visible Light
Ammathnadu S. Amrutha, K. R. Sunil Kumar, Takashi Kikukawa, Nobuyuki Tamaoki
ACS Nano, 11, 12, 12292, 12301, American Chemical Society, 2017年12月26日, ［査読有り］
英語, 研究論文（学術雑誌）

Enhanced expression of cysteine-rich antimicrobial peptide snakin-1 in Escherichia coli using an aggregation-prone protein coexpression system
Md. Ruhul Kuddus, Megumi Yamano, Farhana Rumi, Takashi Kikukawa, Makoto Demura, Tomoyasu Aizawa
BIOTECHNOLOGY PROGRESS, 33, 6, 1520, 1528, 2017年11月, ［査読有り］
英語, 研究論文（学術雑誌）

Spontaneous stacking of purple membranes during immobilization with physical cross-linked poly(vinyl alcohol) hydrogel with retaining native-like functionality of bacteriorhodopsin
Yasunori Yokoyama, Hikaru Tanaka, Shunsuke Yano, Hiroshi Takahashi, Takashi Kikukawa, Masashi Sonoyama, Koshi Takenaka
JOURNAL OF APPLIED PHYSICS, 121, 20, 2017年05月, ［査読有り］
英語, 研究論文（学術雑誌）

Transient Resonance Raman Spectroscopy of a Light-Driven Sodium-Ion-Pump Rhodopsin from Indibacter alkaliphilus
Kousuke Kajimoto, Takashi Kikukawa, Hiroki Nakashima, Haruki Yamaryo, Yuta Saito, Tomotsumi Fujisawa, Makoto Demura, Masashi Unno
JOURNAL OF PHYSICAL CHEMISTRY B, 121, 17, 4431, 4437, 2017年05月, ［査読有り］
英語, 研究論文（学術雑誌）

Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics
Takashi Tsukamoto, Susumu Yoshizawa, Takashi Kikukawa, Makoto Demura, Yuki Sudo
JOURNAL OF PHYSICAL CHEMISTRY B, 121, 9, 2027, 2038, 2017年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Existence of two O-like intermediates in the photocycle of Acetabularia rhodopsin II, a light-driven proton pump from a marine alga
Tamogami, J, Kikukawa, T, Nara, T, Demura, M, Kimura-Someya, T, Shirouzu, M, Yokoyama, S, Miyauchi, S, Shimono, K, Kamo, N
Biophysics and Physicobiology, 14, 49, 55, 2017年, ［査読有り］, ［国内誌］
英語, 研究論文（学術雑誌）, A spectrally silent change is often observed in the photocycle of microbial rhodopsins. Here, we suggest the presence of two O intermediates in the photocycle of Acetabularia rhodopsin II (ARII or also called Ace2), a light-driven algal proton pump from Acetabularia acetabulum. ARII exhibits a photocycle including a quasi-equilibrium state of M, N, and O (M⇄N⇄O→) at near neutral and above pH values. However, acidification of the medium below pH ~5.5 causes no accumulation of N, resulting in that the photocycle of ARII can be described as an irreversible scheme (M→O→). This may facilitate the investigation of the latter part of the photocycle, especially the rise and decay of O, during which molecular events have not been sufficiently understood. Thus we analyzed the photocycle under acidic conditions (pH ≤ 5.5). Analysis of the absorbance change at 610 nm, which mainly monitors the fractional concentration changes of K and O, was performed and revealed a photocycle scheme containing two sequential O-states with the different molar extinction coefficients. These photoproducts, termed O1 and O2, may be even produced at physiological pH, although they are not clearly observed under this condition due to the existence of a long M-N-O equilibrium.

Photochemical characterization of actinorhodopsin and its functional existence in the natural host
Shintaro Nakamura, Takashi Kikukawa, Jun Tamogami, Masakatsu Kamiya, Tomoyasu Aizawa, Martin W. Hahn, Kunio Ihara, Naoki Kamo, Makoto Demura
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 1857, 12, 1900, 1908, 2016年12月, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris
Md. Ruhul Kuddus, Farhana Rumi, Motosuke Tsutsumi, Rika Takahashi, Megumi Yamano, Masakatsu Kamiya, Takashi Kikukawa, Makoto Demura, Tomoyasu Aizawa
PROTEIN EXPRESSION AND PURIFICATION, 122, 15, 22, 2016年06月, ［査読有り］
英語, 研究論文（学術雑誌）

Lipopolysaccharide-bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy
Mi-Hwa Baek, Masakatsu Kamiya, Takahiro Kushibiki, Taichi Nakazumi, Satoshi Tomisawa, Chiharu Abe, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Keiichi Kawano, Tomoyasu Aizawa
JOURNAL OF PEPTIDE SCIENCE, 22, 4, 214, 221, 2016年04月, ［査読有り］
英語, 研究論文（学術雑誌）

In vivo fluorescence correlation spectroscopy analyses of FMBP-1, a silkworm transcription factor
Motosuke Tsutsumi, Hideki Muto, Shohei Myoba, Mai Kimoto, Akira Kitamura, Masakatsu Kamiya, Takashi Kikukawa, Shigeharu Takiya, Makoto Demura, Keiichi Kawano, Masataka Kinjo, Tomoyasu Aizawa
FEBS OPEN BIO, 6, 2, 106, 125, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Formation of M-Like Intermediates in Proteorhodopsin in Alkali Solutions (pH ≥ ∼8.5) Where the Proton Release Occurs First in Contrast to the Sequence at Lower pH.
Tamogami J, Sato K, Kurokawa S, Yamada T, Nara T, Demura M, Miyauchi S, Kikukawa T, Muneyuki E, Kamo N
Biochemistry, 55, 7, 1036, 1048, 2016年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Structure determination of uniformly C-13, N-15 labeled protein using qualitative distance restraints from MAS solid-state C-13-NMR observed paramagnetic relaxation enhancement
Tamaki, Hajime, Egawa, Ayako, Kido, Kouki, Kameda, Tomoshi, Kamiya, Masakatsu, Kikukawa, Takashi, Aizawa, Tomoyasu, Fujiwara, Toshimichi, Demura, Makoto
JOURNAL OF BIOMOLECULAR NMR, 64, 1, 87, 101, 2016年01月, ［査読有り］
英語, 研究論文（学術雑誌）

Characterization of a Cyanobacterial Chloride-pumping Rhodopsin and Its Conversion into a Proton Pump
Takatoshi Hasemi, Takashi Kikukawa, Naoki Kamo, Makoto Demura
JOURNAL OF BIOLOGICAL CHEMISTRY, 291, 1, 355, 362, 2016年01月, ［査読有り］, ［責任著者］
英語, 研究論文（学術雑誌）

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum
Munenori Furuse, Jun Tamogami, Toshiaki Hosaka, Takashi Kikukawa, Naoko Shinya, Masakatsu Hato, Noboru Ohsawa, So Young Kim, Kwang-Hwan Jung, Makoto Demura, Seiji Miyauchi, Naoki Kamo, Kazumi Shimono, Tomomi Kimura-Someya, Shigeyuki Yokoyama, Mikako Shirouzu
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 71, 2203, 2216, 2015年11月, ［査読有り］
英語, 研究論文（学術雑誌）

Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization.
Tomisawa S, Sato Y, Kamiya M, Kumaki Y, Kikukawa T, Kawano K, Demura M, Nakamura K, Ayabe T, Aizawa T
Protein expression and purification, 112, 21, 28, 2015年08月, ［査読有り］
英語, 研究論文（学術雑誌）

Probing the Cl--pumping photocycle of pharaonis halorhodopsin: Examinations with bacterioruberin, an intrinsic dye, and membrane potential-induced modulation of the photocycle
Takashi Kikukawa, Chikara Kusakabe, Asami Kokubo, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Kunio Ihara, Naoki Kamo, Makoto Demura
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 1847, 8, 748, 758, 2015年08月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Microbial rhodopsins of Halorubrum species isolated from Ejinoor salt lake in Inner Mongolia of China
Chaoluomeng, Gang Dai, Takashi Kikukawa, Kunio Ihara, Tatsuo Iwasa
PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 14, 11, 1974, 1982, 2015年, ［査読有り］
英語, 研究論文（学術雑誌）

The effects of chloride ion binding on the photochemical properties of sensory rhodopsin II from Natronomonas pharaonis
Jun Tamogami, Katsunori Iwano, Atsushi Matsuyama, Takashi Kikukawa, Makoto Demura, Toshifumi Nara, Naoki Kamo
JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY, 141, 192, 201, 2014年12月, ［査読有り］
英語, 研究論文（学術雑誌）

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide.
Kushibiki T, Kamiya M, Aizawa T, Kumaki Y, Kikukawa T, Mizuguchi M, Demura M, Kawabata S, Kawano K
Biochimica et biophysica acta, 1844, 3, 527, 534, 2014年03月, ［査読有り］
研究論文（学術雑誌）

Role of Thr218 in the Light-Driven Anion Pump Halorhodopsin from Natronomonas pharaonis
Kousuke Shibasaki, Hiroaki Shigemura, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
BIOCHEMISTRY, 52, 51, 9257, 9268, 2013年12月, ［査読有り］
英語, 研究論文（学術雑誌）

Physicochemical Studies of Bacteriorhodopsin Reconstituted in Partially Fluorinated Phosphatidylcholine Bilayers
Masaru Yoshino, Takashi Kikukawa, Hiroshi Takahashi, Toshiyuki Takagi, Yasunori Yokoyama, Hideki Amii, Teruhiko Baba, Toshiyuki Kanamori, Masashi Sonoyama
JOURNAL OF PHYSICAL CHEMISTRY B, 117, 18, 5422, 5429, 2013年05月, ［査読有り］
英語, 研究論文（学術雑誌）

Salt bridge in the conserved His-Asp cluster in Gloeobacter rhodopsin contributes to trimer formation
Takashi Tsukamoto, Takashi Kikukawa, Takuro Kurata, Kwang-Hwan Jung, Naoki Kamo, Makoto Demura
FEBS Letters, 587, 4, 322, 327, 2013年02月14日, ［査読有り］
英語, 研究論文（学術雑誌）

Exploring the Active Site Structure of a Photoreceptor Protein by Raman Optical Activity
Masashi Unno, Takashi Kikukawa, Masato Kumauchi, Naoki Kamo
JOURNAL OF PHYSICAL CHEMISTRY B, 117, 5, 1321, 1325, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Thermodynamic parameters of anion binding to halorhodopsin from Natronomonas pharaonis by isothermal titration calorimetry
Saori Hayashi, Jun Tamogami, Takashi Kikukawa, Haruka Okamoto, Kazumi Shimono, Seiji Miyauchi, Makoto Demura, Toshifumi Nara, Naoki Kamo
BIOPHYSICAL CHEMISTRY, 172, 61, 67, 2013年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system
Satoshi Tomisawa, Eri Hojo, Yoshitaka Umetsu, Shinya Ohki, Yusuke Kato, Mitsuhiro Miyazawa, Mineyuki Mizuguchi, Masakatsu Kamiya, Yasuhiro Kumaki, Takashi Kikukawa, Keiichi Kawano, Makoto Demura, Tomoyasu Aizawa
AMB EXPRESS, 3, 1, 8, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）

A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method
Satoshi Tomisawa, Chiharu Abe, Masakatsu Kamiya, Takashi Kikukawa, Makoto Demura, Keiichi Kawano, Tomoyasu Aizawa
Biophysics (Japan), 9, 79, 83, 2013年, ［査読有り］
英語, 研究論文（学術雑誌）

Influence of Halide Binding on the Hydrogen Bonding Network in the Active Site of Salinibacter Sensory Rhodopsin I
Louisa Reissig, Tatsuya Iwata, Takashi Kikukawa, Makoto Demura, Naoki Kamo, Hideki Kandori, Yuki Sudo
BIOCHEMISTRY, 51, 44, 8802, 8813, 2012年11月, ［査読有り］
英語, 研究論文（学術雑誌）

Photoinduced Proton Release in Proteorhodopsin at Low pH: The Possibility of a Decrease in the pK(a) of Asp227
Jun Tamogami, Takashi Kikukawa, Toshifumi Nara, Kazumi Shimono, Makoto Demura, Naoki Kamo
BIOCHEMISTRY, 51, 46, 9290, 9301, 2012年11月, ［査読有り］
英語, 研究論文（学術雑誌）

Dynamics of Dangling Bonds of Water Molecules in pharaonis Halorhodopsin during Chloride Ion Transportation
Yuji Furutani, Kuniyo Fujiwara, Tetsunari Kimura, Takashi Kikukawa, Makoto Demura, Hideki Kandori
JOURNAL OF PHYSICAL CHEMISTRY LETTERS, 3, 20, 2964, 2969, 2012年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System
Takashi Tsukamoto, Takanori Sasaki, Kazuhiro J. Fujimoto, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
BIOPHYSICAL JOURNAL, 102, 12, 2906, 2915, 2012年06月, ［査読有り］
英語, 研究論文（学術雑誌）

Photo-induced bleaching of sensory rhodopsin II (phoborhodopsin) from Halobacterium salinarum by hydroxylamine: Identification of the responsible intermediates
Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Makoto Demura, Toshifumi Nara, Naoki Kamo
JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY, 106, 1, 87, 94, 2012年01月, ［査読有り］
英語, 研究論文（学術雑誌）

Conststruction of a novel expression system for cryptdin-4 by using inclusion body formation.　　　　　　　　　　　　　　　
Sato Y, Tomisawa S, Aizawa T, Sakai N, Kamiya M, Kikukawa T, Kumaki Y, Demura M, Ayabe T, Kawano K
Peptide Science 2011, 393, 394, 2012年, ［査読有り］

Expression of salinarum halorhodopsin in Escherichia coli cells: solubilization in the presence of retinal yields the natural state.
Yamashita Y, Kikukawa T, Tsukamoto T, Kamiya M, Aizawa T, Kawano K, Miyauchi S, Kamo N, Demura M
Biochimica et biophysica acta, 1808, 12, 2905, 2912, 2011年12月, ［査読有り］, ［責任著者］
研究論文（学術雑誌）

Photochemistry of Acetabularia Rhodopsin II from a Marine Plant, Acetabularia acetabulum
Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Seiji Miyauchi, So Young Kim, Tomomi Kimura-Someya, Mikako Shirouzu, Kwang-Hwan Jung, Shigeyuki Yokoyama, Naoki Kamo
BIOCHEMISTRY, 50, 41, 8888, 8898, 2011年10月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Crystal Structure of the Eukaryotic Light-Driven Proton-Pumping Rhodopsin, Acetabularia Rhodopsin II, from Marine Alga
Takashi Wada, Kazumi Shimono, Takashi Kikukawa, Masakatsu Hato, Naoko Shinya, So Young Kim, Tomomi Kimura-Someya, Mikako Shirouzu, Jun Tamogami, Seiji Miyauchi, Kwang-Hwan Jung, Naoki Kamo, Shigeyuki Yokoyama
JOURNAL OF MOLECULAR BIOLOGY, 411, 5, 986, 998, 2011年09月, ［査読有り］
英語, 研究論文（学術雑誌）

Spectroscopic Evidence for the Formation of an N Intermediate during the Photocycle of Sensory Rhodopsin II (Phoborhodopsin) from Natronobacterium pharaonis
Yusuke Tateishi, Takayuki Abe, Jun Tamogami, Yutaka Nakao, Takashi Kikukawa, Naoki Kamo, Masashi Unno
BIOCHEMISTRY, 50, 12, 2135, 2143, 2011年03月, ［査読有り］
英語, 研究論文（学術雑誌）

A Microbial Rhodopsin with a Unique Retinal Composition Shows Both Sensory Rhodopsin II and Bacteriorhodopsin-like Properties
Yuki Sudo, Kunio Ihara, Shiori Kobayashi, Daisuke Suzuki, Hiroki Irieda, Takashi Kikukawa, Hideki Kandori, Michio Homma
JOURNAL OF BIOLOGICAL CHEMISTRY, 286, 8, 5967, 5976, 2011年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Photochemistry of a putative new class of sensory rhodopsin (SRIII) coded by xop2 of Haloarcular marismortui
Yutaka Nakao, Takashi Kikukawa, Kazumi Shimono, Jun Tamogami, Noriko Kimitsuki, Toshifumi Nara, Masashi Unno, Kunio Ihara, Naoki Kamo
JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY, 102, 1, 45, 54, 2011年01月, ［査読有り］
英語, 研究論文（学術雑誌）

ＳｎＯ２透明電極を用いた光受容タンパク質のプロトン移動解析
田母神 淳, 菊川 峰志
生物物理, 51, 1, 042, 043, 一般社団法人 日本生物物理学会, 2011年, ［査読有り］, ［最終著者］
日本語

STPR, a 23-Amino Acid Tandem Repeat Domain, Found in the Human Function-Unknown Protein ZNF821
Yasuhiro Nonaka, Hideki Muto, Tomoyasu Aizawa, Etsuro Okabe, Shohei Myoba, Takuya Yokoyama, Shin Saito, Fumie Tatami, Yasuhiro Kumaki, Masakatsu Kamiya, Takashi Kikukawa, Mineyuki Mizuguchi, Shigeharu Takiya, Masataka Kinjo, Makoto Demura, Keiichi Kawano
BIOCHEMISTRY, 49, 38, 8367, 8375, 2010年09月, ［査読有り］
英語, 研究論文（学術雑誌）

Photoreaction Cycle of Phoborhodopsin (Sensory Rhodopsin II) from Halobacterium salinarum Expressed in Escherichia coli
Gang Dai, Yoshikazu Ohno, Yoichi Ikeda, Jun Tamogami, Takashi Kikukawa, Naoki Kamo, Tatsuo Iwasa
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 86, 3, 571, 579, 2010年05月, ［査読有り］
英語, 研究論文（学術雑誌）

The Photochemical Reaction Cycle and Photoinduced Proton Transfer of Sensory Rhodopsin II (Phoborhodopsin) from Halobacterium salinarum
Jun Tamogami, Takashi Kikukawa, Yoichi Ikeda, Ayaka Takemura, Makoto Demura, Naoki Kamo
BIOPHYSICAL JOURNAL, 98, 7, 1353, 1363, 2010年04月, ［査読有り］
英語, 研究論文（学術雑誌）

Kinetic study of anti-viral ribavirin uptake mediated by hCNT3 and hENT1 in Xenopus laevis oocytes
Takashi Yamamoto, Mitsuru Sugawara, Takashi Kikukawa, Seiji Miyauchi, Masahiro Yamaguchi, Atsushi Tero, Seiji Takagi, Toshiyuki Nakagaki
BIOPHYSICAL CHEMISTRY, 147, 1-2, 59, 65, 2010年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Spectroscopic Studies of a Sensory Rhodopsin I Homologue from the Archaeon Haloarcula vallismortis
Jin Yagasaki, Daisuke Suzuki, Kunio Ihara, Keiichi Inoue, Takashi Kikukawa, Makoto Sakai, Masaaki Fujii, Michio Homma, Hideki Kandori, Yuki Sudo
BIOCHEMISTRY, 49, 6, 1183, 1190, 2010年02月, ［査読有り］
英語, 研究論文（学術雑誌）

Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I (vol 392, pg 48, 2009)
Suzuki, Daisuke, Furutani, Yuji, Inoue, Keiichi, Kikukawa, Takashi, Sakai, Makoto, Fujii, Masaaki, K, ori, Hideki, Homma, Michio, Sudo, Yuki
Journal of Molecular Biology, 395, 1, 2010年
研究論文（学術雑誌）

Effect of Molecular Assembly on Photocycle of Reconstituted Bacteriorhodopsin: Significant Blue Shift of the Late M Photointermediate in the Liquid Crystalline Phase
Masashi Sonoyama, Takashi Kikukawa, Yasunori Yokoyama, Makoto Demura, Naoki Kamo, Shigeki Mitaku
CHEMISTRY LETTERS, 38, 12, 1134, 1135, 2009年12月, ［査読有り］
英語, 研究論文（学術雑誌）

Effect of Chloride Binding on the Thermal Trimer-Monomer Conversion of Halorhodopsin in the Solubilized System
Takanori Sasaki, Tomoyasu Aizawa, Masakatsu Kamiya, Takashi Kikukawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
BIOCHEMISTRY, 48, 51, 12089, 12095, 2009年12月, ［査読有り］
英語, 研究論文（学術雑誌）

Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid
Kazumi Shimono, Mie Goto, Takashi Kikukawa, Seiji Miyauchi, Mikako Shirouzu, Naoki Kamo, Shigeyuki Yokoyama
PROTEIN SCIENCE, 18, 10, 2160, 2171, 2009年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Effects of Chloride Ion Binding on the Photochemical Properties of Salinibacter Sensory Rhodopsin I
Daisuke Suzuki, Yuji Furutan, Keiichi Inoue, Takashi Kikukawa, Makoto Sakai, Masaaki Fujii, Hideki Kandori, Michio Homma, Yuki Sudo
JOURNAL OF MOLECULAR BIOLOGY, 392, 1, 48, 62, 2009年09月, ［査読有り］
英語, 研究論文（学術雑誌）

X-ray crystallography and structural stability of digestive lysozyme from cow stomach
Yasuhiro Nonaka, Daisuke Akieda, Tomoyasu Aizawa, Nobuhisa Watanabe, Masakatsu Kamiya, Yasuhiro Kumaki, Mineyuki Mizuguchi, Takashi Kikukawa, Makoto Demura, Keiichi Kawano
FEBS JOURNAL, 276, 8, 2192, 2200, 2009年04月, ［査読有り］
英語, 研究論文（学術雑誌）

A Tin Oxide Transparent Electrode Provides the Means for Rapid Time-resolved pH Measurements: Application to Photoinduced Proton Transfer of Bacteriorhodopsin and Proteorhodopsin
Jun Tamogami, Takashi Kikukawa, Seiji Miyauchi, Eiro Muneyuki, Naoki Kamo
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 85, 2, 578, 589, 2009年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Role of Arg123 in Light-driven Anion Pump Mechanisms of pharaonis Halorhodopsin
Megumi Kubo, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 85, 2, 547, 555, 2009年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Halorhodopsin from Natronomonas pharaonis Forms a Trimer Even in the Presence of a Detergent, Dodecyl-beta-d-maltoside
Takanori Sasaki, Megumi Kubo, Takashi Kikukawa, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Naoki Kamo, Makoto Demura
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 85, 1, 130, 136, 2009年01月, ［査読有り］
英語, 研究論文（学術雑誌）

Heat-treatment method for producing fatty acid-bound alpha-lactalbumin that induces tumor cell death
Tatsuro Kamijima, Ayaka Ohmura, Toshiya Sato, Kaoru Akimoto, Miki Itabashi, Mineyuki Mizuguchi, Masakatsu Kamiya, Takashi Kikukawa, Tomoyasu Aizawa, Masayuki Takahashi, Keiichi Kawano, Makoto Demura
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 376, 1, 211, 214, 2008年11月, ［査読有り］
英語, 研究論文（学術雑誌）

The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers - Effects of hydrostatic pressure and stopped flow experiments
Takashi Kikukawa, Chabita K. Saha, Sergei P. Balashov, Eleonora S. Imasheva, Dmitry Zaslavsky, Robert B. Gennis, Takayuki Abe, Naoki Kamo
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 84, 4, 880, 888, 2008年07月, ［査読有り］, ［筆頭著者］
英語, 研究論文（学術雑誌）

Anti-parallel membrane topology of a homo-dimeric multidrug transporter, EmrE
Toshifumi Nara, Tomoko Kouyama, Yuko Kurata, Takashi Kikukawa, Seiji Miyauchi, Naoki Kamo
JOURNAL OF BIOCHEMISTRY, 142, 5, 621, 625, 2007年11月, ［査読有り］
英語, 研究論文（学術雑誌）

Anti-parallel membrane topology of two components of EbrAB, a multidrug transporter
Takashi Kikukawa, Seiji Miyauchi, Tsunehisa Araiso, Naoki Kamo, Toshifumi Nara
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 358, 4, 1071, 1075, 2007年07月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Heterologous expression of Pharaonis halorhodopsin in Xenopus laevis oocytes and electrophysiological characterization of its light-driven C- pump activity
Akiteru Seki, Seiji Miyauchi, Saori Hayashi, Takashi Kikukawa, Megumi Kubo, Makoto Demura, Vadivel Ganapathy, Naoki Kamo
BIOPHYSICAL JOURNAL, 92, 7, 2559, 2569, 2007年04月, ［査読有り］
英語, 研究論文（学術雑誌）

Interaction of the halobacterial transducer to a halorhodopsin mutant engineered so as to bind the transducer: Cl- circulation within the extracellular channel
Chisa Hasegawa, Takashi Kikukawa, Seiji Miyauchi, Akiteru Seki, Yuki Sudo, Megumi Kubo, Makoto Demura, Naoki Kamo
PHOTOCHEMISTRY AND PHOTOBIOLOGY, 83, 2, 293, 302, 2007年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Difference between Pharmacokinetics of Mycophenolic acid (MPA) in Rats and that in Humans is caused by Different affinities of MRP2 to a glucuronized form
Yoh Takekuma, Haruka Kakiuchi, Koujiro Yamazaki, Seiji Miyauchi, Takashi Kikukawa, Naoki Kamo, Vadivel Ganapathy, Mitsuru Sugawara
JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES, 10, 1, 71, 85, 2007年01月, ［査読有り］
英語, 研究論文（学術雑誌）

ヘテロダイマー型ＳＭＲ：ＥｂｒＡＢの膜内トポロジーの決定とモノマー単独での機能発現
菊川 峰志, 奈良 敏文
生物物理, 47, 4, 264, 267, 一般社団法人 日本生物物理学会, 2007年, ［査読有り］, ［筆頭著者, 責任著者］
日本語

All-optical switching in pharaonis phoborhodopsin protein molecules
Sukhdev Roy, Takashi Kikukawa, Parag Sharma, Naoki Kamo
IEEE TRANSACTIONS ON NANOBIOSCIENCE, 5, 3, 178, 187, 2006年09月, ［査読有り］
英語, 研究論文（学術雑誌）

Two-component bacterial multidrug transporter, EbrAB: Mutations making each component solely functional
Takashi Kikukawa, Toshifumi Nara, Tsunehisa Araiso, Seiji Miyauchi, Naoki Kamo
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1758, 5, 673, 679, 2006年05月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

A light-driven proton pump from Haloterrigena turkmenica: Functional expression in Escherichia coli membrane and coupling with a H+ co-transporter
N Kamo, T Hashiba, T Kikukawa, T Araiso, K Ihara, T Nara
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 341, 2, 285, 290, 2006年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Role of putative anion-binding sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin
M Sato, M Kubo, T Aizawa, N Kamo, T Kikukawa, K Nitta, M Demura
BIOCHEMISTRY, 44, 12, 4775, 4784, 2005年03月, ［査読有り］
英語, 研究論文（学術雑誌）

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding
M Sato, T Kikukawa, T Araiso, H Okita, K Shimono, N Kamo, M Demura, K Nitta
BIOPHYSICAL CHEMISTRY, 104, 1, 209, 216, 2003年05月, ［査読有り］
英語, 研究論文（学術雑誌）

Changes in lipid mobility associated with alamethicin incorporation into membranes
T Kikukawa, T Araiso
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 405, 2, 214, 222, 2002年09月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Pharmacological characterization of emotional responses to unpredictable microgravity conditions
M Yoshioka, S Ohashi, T Kikukawa, T Araiso, S Yano, M Tamura
JAPANESE JOURNAL OF PHARMACOLOGY, 88, 234P, 234P, 2002年, ［査読有り］
英語

Effects of anxiolytics on hemodynamic responses to acute microgravity environment
Yoshioka, M, Kikukawa, T, Ohashi, S, Araiso, T, Yano, S, Tamura, M
Biological Sciences in Space, 14, 3, 190, 191, 2000年10月, ［査読有り］
英語, 研究論文（学術雑誌）

Polymerization of pyrrole in microgravity
T Nakamura, T Akutagawa, T Hasegawa, T Kikukawa, T Araiso, M Higuchi, K Hiratani
SYNTHETIC METALS, 101, 1-3, 78, 79, 1999年05月, ［査読有り］
英語, 研究論文（学術雑誌）

Azide accelerates the decay of M-intermediate of pharaonis phoborhodopsin
K Takao, T Kikukawa, T Araiso, N Kamo
BIOPHYSICAL CHEMISTRY, 73, 1-2, 145, 153, 1998年07月, ［査読有り］
英語, 研究論文（学術雑誌）

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol
T Kikukawa, T Araiso, T Shimozawa, K Mukasa, N Kamo
BIOPHYSICAL JOURNAL, 73, 1, 357, 366, 1997年07月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Culture temperature affects the molecular motion of bacteriorhodopsin within the purple membrane
Takashi Kikukawa, Tsunchisa Araiso, Kôichi Mukasa, Tateo Shimozawa, Naoki Kamo
Chemical and Pharmaceutical Bulletin, 44, 3, 473, 476, Pharmaceutical Society of Japan, 1996年, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane
T Kikukawa, T Araiso, K Mukasa, T Shimozawa, N Kamo
FEBS LETTERS, 377, 3, 502, 504, 1995年12月, ［査読有り］, ［筆頭著者, 責任著者］
英語, 研究論文（学術雑誌）

Discovery of Light-Powered Organic Ion Transport by a Natural Protein
Simiao Shen, Shoichiro Akita, Joji Wada, Mako Eguchi, Takashi Tsukamoto, Kwang-Hwan Jung, Yuki Sudo, Takashi Kikukawa, bioRxiv, 2025年07月04日, ［最終著者, 責任著者］
ABSTRACT

Membrane transport proteins play vital roles in living cells by selectively transporting ions and molecules across biological membranes. Among these, microbial rhodopsins are unique in their unparalleled capacity to harness light to drive ion translocation. This distinctive feature has led to their widespread use as optogenetic tools in neuroscience, physiology, and biomed-ical applications. While not all microbial rhodopsins function as ion transporters, many ion-translocating variants have been discov-ered since the identification of the first member—a light-driven H⁺ pump—in the 1970s. These proteins share a compact structure composed of only seven transmembrane helices and have long been thought to specialize exclusively in transporting small inorganic ions such as H⁺, Cl⁻, and Na⁺. Here, we show that several anion-pumping microbial rhodopsins can also transport organic anions. In particular, a rhodopsin from cyanobacteria is capable of transporting bulky organic anions, including those containing benzene rings, with molecular volumes up to ∼120 ų—five times that of Cl⁻. These organic ions bind to the dark state and are translocated upon photoactivation, following a mechanism similar to that of inorganic anion transport. Mutational analysis indicates that both classes of substrates share a common binding site. Only anions with pKa values below 2 were transported, suggesting that a retained negative charge is essential for binding to the dark state—a prerequisite for transport. This study expands the known sub-strate repertoire of microbial rhodopsins and introduces new possibilities for optogenetic strategies based on light-driven delivery of bioactive organic molecules., Cold Spring Harbor Laboratory

カルモジュリン融合タンパク質システムを使用した安定同位体標識セクロピンP1の大量発現とNMR構造解析
GU Hao, 加藤貴純, 石田博昭, 熊木康裕, 塚本卓, 塚本卓, 菊川峰志, 菊川峰志, 出村誠, 出村誠, VOGEL HANS J., 相沢智康, 相沢智康, Abstracts. Annual Meeting of the NMR Society of Japan, 59th, 2020年

Functional significance of interhelical interactions between aspartic acid and cysteine residues in microbial rhodopsin
Jun Tamogami, Takashi Kikukawa, Naoki Kamo, Atlas of Science (Web), 2019年06月
英語, その他

部分フッ素化リン脂質F4-DMPC膜に再構成した膜タンパク質バクテリオロドプシンの2次元格子の安定性
高橋浩, 吉野賢, 森田康平, 高木俊之, 横山泰範, 菊川峰志, 網井秀樹, 金森敏幸, 園山正史, 園山正史, 園山正史, KEK Progress Report (Web), 2019-7, 2019年

擬環状エーテル型脂質のフッ素化が膜物性および膜タンパク質の構造・機能に及ぼす影響
川原るい, 高木俊之, 高橋浩, 菊川峰志, 網井秀樹, 園山正史, 園山正史, 園山正史, フッ素化学討論会講演要旨集, 42nd, 2019年

マウス抗菌ペプチドcryptdin‐4の変異体を用いた作用機構の解析
平峰里菜, 久米田博之, 久米田博之, 熊木康裕, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 中村公則, 綾部時芳, 相沢智康, 相沢智康, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.4J29a01 (WEB ONLY), 2017年03月05日
日本語

ジャガイモ由来抗菌ペプチドsnakin‐1の発現系構築
山野めぐみ, KUDDUS MD Ruhul, RUMI Farhana, 堤元佐, 久米田博之, 久米田博之, 熊木智康, 神谷昌克, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 相沢智康, 相沢智康, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.4J29a04 (WEB ONLY), 2017年03月05日
日本語

シマグワワインのNMRメタボローム解析
稲村勇雅, 久米田博之, 久米田博之, 熊木康裕, 大西裕季, 菊川峰志, 菊川峰志, 出村誠, 出村誠, 小山朗夫, 伊東昌章, 岩波俊介, 相沢智康, 相沢智康, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.3A04p09 (WEB ONLY), 2017年03月05日
日本語

ナトリウムポンプ型ロドプシンにおけるイオン結合サイトの役割
田巻初, 田巻初, 斉藤優太, 江川文子, 菊川峰志, 相沢智康, 出村誠, 藤原敏道, Abstracts. Annual Meeting of the NMR Society of Japan, 55th, 230‐231, 2016年11月16日
日本語

ジャガイモ由来抗菌ペプチドsnakin‐1の安定的な発現・精製系の構築とNMRによる構造解析
山野めぐみ, 神谷昌克, 熊木康裕, 菊川峰志, 出村誠, 相沢智康, 日本農芸化学会大会講演要旨集(Web), 2016, 4C001 (WEB ONLY), 2016年03月05日
日本語

部分フッ素化リン脂質膜に再構成したバクテリオロドプシンが形成する六方格子構造の熱安定性
高橋浩, 吉野賢, 高木俊之, 菊川峰志, 網井秀樹, 金森敏幸, 園山正史, 日本物理学会講演概要集(CD-ROM), 71, 1, 2016年

部分フッ素化リン脂質の膜構造の特徴とその膜に再構成したバクテリオロドプシンの二次元格子の熱安定性
高橋浩, 吉野賢, 高木俊之, 菊川峰志, 網井秀樹, 金森敏幸, 園山正史, KEK Proceedings (Web), 2016-2, 2016年

新規部分フッ素化リン脂質ライブラリーの構築:膜タンパク質バクテリオロドプシン研究への応用
吉野賢, 橋本麻美, 堀越未希, 村井裕佳, 森田康平, 柳瑶美, 茂木俊憲, 菊川峰志, 高木俊之, 高橋浩, 網井秀樹, 金森敏幸, 園山正史, フッ素化学討論会講演要旨集, 39th, 2016年

固体NMRによるタンパク質測定への圧縮センシングの応用
田巻初, 斉藤優太, 江川文子, 菊川峰志, 神谷昌克, 相沢智康, 藤原敏道, 出村誠, Abstracts. Annual Meeting of the NMR Society of Japan, 54th, 236, 237, 2015年11月06日
日本語

抗菌ペプチドthanatinとリポ多糖の相互作用解析
若松瞭太, 神谷昌克, 相沢智康, 熊木康裕, 菊川峰志, 出村誠, Abstr Annu Meet NMR Soc Jpn, 54th, 156, 157, 2015年11月06日
日本語

固体NMR法を用いたナトリウムポンプ製ロドプシンの構造解析
斉藤優太, 田巻初, 江川文子, 菊川峰志, 神谷昌克, 相沢智康, 藤原敏道, 出村誠, Abstr Annu Meet NMR Soc Jpn, 54th, 234, 235, 2015年11月06日
日本語

部分フッ素化リン脂質を利用した膜タンパク質研究の展開
園山正史, 吉野賢, 村井裕佳, 森田康平, 菊川峰志, 高木俊之, 高橋浩, 網井秀樹, 網井秀樹, 金森敏幸, 日本蛋白質科学会年会プログラム・要旨集, 15th, 2015年

疎水鎖末端をパーフルオロアルキル基に置換したDMPC脂質二分子膜中の膜タンパク質の構造・機能・安定性 2.DMPCを含む二成分系混合膜におけるバクテリオロドプシンの分配挙動
堀越未希, 吉野賢, 金山賢治, 森田康平, 高橋浩, 網井秀樹, 菊川峰志, 高木俊之, 金森敏幸, 園山正史, 分子科学討論会講演プログラム&要旨(Web), 9th, 2015年

疎水鎖末端をパーフルオロアルキル基に置換したDMPC脂質二分子膜中の膜タンパク質の構造・機能・安定性 1.パーフルオロアルキル基によるバクテリオロドプシンの安定化
橋本麻美, 村井裕佳, 森田康平, 高橋浩, 網井秀樹, 菊川峰志, 高木俊之, 金森敏幸, 園山正史, 分子科学討論会講演プログラム&要旨(Web), 9th, 2015年

高圧閃光光分解法を用いた光駆動性アニオンポンプ:ハロロドプシンのCl^-輸送機構の解析
柴崎宏介, 重村洋明, 菊川峰志, 神谷昌克, 相沢智康, 河野敬一, 加茂直樹, 出村誠, 高圧討論会講演要旨集, 55th, 71, 2014年11月10日
日本語

膜タンパク質ASRとの相互作用解析に向けたASRTのNMR解析
比嘉一葉, 神谷昌克, 菊川峰志, 熊木康裕, 出村誠, 相沢智康, Abstr Annu Meet NMR Soc Jpn, 53rd, 234, 235, 2014年11月04日
日本語

常磁性緩和促進を利用したDNA結合ドメインSTPRの分子ダイナミクスの解析
柚原光佑, 神谷昌克, 熊木康裕, 滝谷重治, 菊川峰志, 河野敬一, 出村誠, 相沢智康, Abstr Annu Meet NMR Soc Jpn, 53rd, 244, 245, 2014年11月04日
日本語

固体NMRにおける常磁性緩和促進を用いたタンパク質立体構造解析
田巻初, 江川文子, 木戸浩貴, 亀田倫史, 神谷昌克, 菊川峰志, 相沢智康, 藤原敏道, 出村誠, Abstr Annu Meet NMR Soc Jpn, 53rd, 298, 299, 2014年11月04日
日本語

再構成無細胞系を用いた短鎖ペプチドの直接発現
冨澤聡, 神谷昌克, 菊川峰志, 出村誠, 相沢智康, 日本蛋白質科学会年会プログラム・要旨集, 14th, 145, 2014年05月26日
日本語

二量体化したDi-O-Tetradecylphosphatidylcholineへの膜タンパク質バクテリオロドプシンの再構成
土田直之, 高木俊之, 菊川峰志, 高橋浩, 金森敏幸, 園山正史, 日本化学会講演予稿集, 94th, 3, 2014年

固体NMRにおけるGFT NMRを応用したタンパク質連鎖帰属法ならびに解析支援プログラムの開発
田巻初, 江川文子, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠, Abstr Annu Meet NMR Soc Jpn, 52nd, 272, 273, 2013年11月12日
日本語

固体NMRによるタンパク質の構造解析に向けた¹³C‐常磁性緩和促進の研究
木戸浩貴, 田巻初, 江川文子, 亀田倫史, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠, Abstr Annu Meet NMR Soc Jpn, 52nd, 286, 287, 2013年11月12日
日本語

部分フッ素化リン脂質を含む二成分系リン脂質混合膜における膜タンパク質バクテリオロドプシンの分配挙動
吉野賢, 金山賢治, 高橋浩, 高木俊之, 菊川峰志, 横山泰範, 網井秀樹, 金森敏之, 園山正史, 生体分子科学討論会講演要旨集, 40th, 84, 85, 2013年06月07日
日本語

CD and NMR Analysis of a Peptide Mimicking the First Extracellular Loop of the Transmembrane Protein Halorhodopsin
TAKANASHI Yoshihiko, KAMIYA Masakatsu, KUMAKI Yasuhiro, KIKUKAWA Takashi, AIZAWA Tomoyasu, KAWANO Keiichi, DEMURA Makoto, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2012, 333, 336, 2013年03月01日
英語

Importance of Salt Bridge in Cryptdin-4 for Folding in Denatured Condition
SATO Yuji, TOMISAWA Satoshi, AIZAWA Tomoyasu, KAMIYA Masakatsu, KIKUKAWA Takashi, KUMAKI Yasuhiro, DEMURA Makoto, KAWANO Keiichi, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2012, 209, 212, 2013年03月01日
英語

NMR Structural Analysis of Potato Antimicrobial Peptide Snakin-1
TAKAHASHI Rika, KAMIYA Masakatsu, AIZAWA Tomoyasu, KUMAKI Yasuhiro, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2012, 51, 54, 2013年03月01日
英語

部分フッ素化リン脂質・非フッ素化リン脂質二成分混合膜に再構成したバクテリオロドプシンの脂質選択性
吉野賢, 菊川峰志, 高橋浩, 高橋浩, 高木俊之, 網井秀樹, 網井秀樹, 金森敏幸, 園山正史, 園山正史, 日本蛋白質科学会年会プログラム・要旨集, 13th, 2013年

部分フッ素化リン脂質膜中に再構成したバクテリオロドプシンの光サイクルと二次元格子構造について
高橋浩, 吉野賢, 菊川峰志, 高木俊之, 横山泰範, 網井秀樹, 馬場照彦, 金森敏幸, 園山正史, 物構研サイエンスフェスタ要旨集, 1st, 57, 2013年
日本語

牛α‐スペクトリンの遺伝子多型(E91K置換)による分子構造と赤血球膜物性の異常
木崎皓太, 富張瑞樹, 大津航, 新敷信人, 塚本卓, 宮園耕介, 菊川峰志, 出村誠, 大塚弥生, 佐藤耕太, 高桑雄一, 稲葉睦, 日本獣医学会学術集会講演要旨集, 154th, 338, 2012年08月31日
日本語

カブトガニ由来抗菌ペプチド Tachyplesin I のキチン結合能
櫛引 崇弘, 神谷 昌克, 相沢 智康, 境 勝義, 石川 和裕, 東 乙比古, 熊木 康裕, 菊川 峰志, 出村 誠, 川端 俊一郎, 河野 敬一, キチン・キトサン研究 = Chitin and chitosan research, 18, 2, 184, 184, 2012年07月01日
日本語

新規部分フッ素化リン脂質に再構成したバクテリオロドプシンに対する脂質フッ素化の影響
吉野賢, 高木俊之, 菊川峰志, 高橋浩, 馬場照彦, 金森敏之, 園山正史, 生体分子科学討論会講演要旨集, 39th, 38, 39, 2012年06月08日
日本語

ハロロドプシンの光反応サイクル中に起こるカロテノイドの吸光度変化
菊川峰志, 小久保麻実, 塚本卓, 井原邦夫, 加茂直樹, 出村誠, 日本蛋白質科学会年会プログラム・要旨集, 12th, 130, 2012年05月31日
日本語

牛α‐スペクトリン・アレルSpαBK91に起因するリピート1の構造異常と赤血球膜物性の変化
木崎皓太, 富張瑞樹, 大塚弥生, 塚本卓, 新敷信人, 菊川峰志, 出村誠, 大津航, 佐藤耕太, 高桑雄一, 稲葉睦, 日本膜学会年会講演要旨集, 34th, 63, 2012年04月27日
日本語

NMR Structure and Interaction of Antimicrobial Peptide Tachyplesin I with Lipopolysaccharide
KUSHIBIKI Takahiro, KAMIYA Masakatsu, AIZAWA Tomoyasu, KUMAKI Yasuhiro, KIKUKAWA Takashi, DEMURA Makoto, KAWABATA Shunichiro, KAWANO Keiichi, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2011, 225, 226, 2012年03月01日
英語

Does Escherichia coli Thioredoxin Improve Expression Efficiency of Recombinant Peptide in Pichia pastoris?
KITAMURA Yuki, AIZAWA Tomoyasu, KAMIYA Masakastu, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2011, 375, 376, 2012年03月01日
英語

Structural Analysis of Plant Antimicrobial Peptide Snakin-1
TAKAHASHI Rika, AIZAWA Tomoyasu, KAMIYA Masakatsu, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2011, 39, 42, 2012年03月01日
英語

Expression and Structural Analysis of a Recombinant Antibacterial Peptide, Cecropin P1
NAKAZUMI Taichi, SAKAI Chihiro, MAEHANA Shiori, AIZAWA Tomoyasu, HOJO Eri, MATSUMOTO Takashi, KAMIYA Masakatsu, KUMAKI Yasuhiro, KIKUKAWA Takashi, DEMURA Makoto, KAWANO Keiichi, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2011, 219, 220, 2012年03月01日
英語

Expression, Purification and NMR Characterization of the Cyclic Reconbinant Form the First Extracellular Loop of the Transmembrane Protein Halorhodopsin
TAKANASHI Yoshihiko, KAMIYA Masakatsu, KUMAKI Yasuhiro, KIKUKAWA Takashi, AIZAWA Tomoyasu, KAWANO Keiichi, DEMURA Makoto, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2011, 223, 224, 2012年03月01日
英語

三次元固体NMR法による膜タンパク質ハロロドプシンの研究
田巻初, 江川文子, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠, 日本蛋白質科学会年会プログラム・要旨集, 12th, 2012年

固体NMRにおけるタンパク質連鎖帰属へのGFT NMR法の応用
田巻初, 江川文子, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠, Abstracts. Annual Meeting of the NMR Society of Japan, 51st, 2012年

Conformational analysis of novel peptide derivatives with cytostatic activity
KAMIYA Masakatsu, YOSHIDA Tomoshi, AIZAWA Tomoyasu, KIKUKAWA Takashi, IMAI Kunio, SUZUKI Koichi, DEMURA Makoto, KAWANO Keiichi, Peptide science : proceedings of the ... Japanese Peptide Symposium, 2010, 160, 160, 2011年03月01日
英語

非線形サンプリングを用いた膜タンパク質の固体NMR測定
田巻初, 江川文子, 松木陽, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 藤原敏道, 出村誠, 日本蛋白質科学会年会プログラム・要旨集, 11th, 2011年

新規微生物型ロドプシン(MR)から観る光駆動イオンポンプから光センサーへの分子進化
須藤雄気, 井原邦夫, 小林詩織, 鈴木大介, 入枝泰樹, 菊川峰志, 神取秀樹, 本間道夫, 生化学, ROMBUNNO.2T16A-2, 2011年
日本語

Exploring the active site structure of photoreceptor proteins by Raman optical activity
Masashi Unno, Takahito Shingae, Takashi Kikukawa, Naoki Kamo, AIP Conference Proceedings, 1267, 102, 103, 2010年12月14日

固体NMR法による7TMハロロドプシン膜外ループの構造解析
田巻初, 樋口真理花, 江川文子, 藤原敏道, 横山順, 横山順, 横山順, 木川隆則, 木川隆則, 下野和実, 下野和実, 染谷友美, 白水美香子, 横山茂之, 横山茂之, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 出村誠, 日本蛋白質科学会年会プログラム・要旨集, 10th, 2010年

膜タンパク質ハロロドプシンの多次元固体NMR-同位体ラベルの最適化-
田巻初, 樋口真理花, 江川文子, 藤原敏道, 横山順, 横山順, 横山順, 木川隆則, 木川隆則, 下野和実, 下野和実, 染谷友美, 白水美香子, 横山茂之, 横山茂之, 神谷昌克, 菊川峰志, 相沢智康, 河野敬一, 出村誠, Abstracts. Annual Meeting of the NMR Society of Japan, 49th, 2010年

2P302 アザイド存在下におけるハロロドプシンのプロトン輸送機構の解析(生体膜・人工膜(輸送),ポスター発表,第45回日本生物物理学会年会)
宮内 正二, 関 顕照, 菊川 峰志, 青山 健太郎, 出村 誠, Ganapathy Vadivel, 加茂 直樹, 生物物理, 47, 1, S188, 2007年11月20日
一般社団法人日本生物物理学会, 英語

3P223 浸透圧を加えた条件下におけるハロロドプシンの光化学反応(光生物(視覚・光受容),ポスター発表,第45回日本生物物理学会年会)
菊川 峰志, 重村 洋明, 久保 恵美, 出村 誠, 宮内 正二, 加茂 直樹, 生物物理, 47, 1, S258, 2007年11月20日
一般社団法人日本生物物理学会, 英語

3P222 光駆動アニオンポンプ・ハロロドプシンのArg123の役割(光生物学(視覚と光受容)、放射線生物学,口頭発表,第45回日本生物物理学会年会)
久保 恵美, 菊川 峰志, 宮内 正二, 関 顕照, 神谷 昌克, 相沢 智康, 河野 敬一, 加茂 直樹, 出村 誠, 生物物理, 47, 1, S258, 2007年11月20日
日本生物物理学会, 英語

1P268 ファラオニスハロロドプシンのクロライド放出側のArgの役割(光生物 A) 視覚・光受容))
佐藤 麻希, 菊川 峰志, 加茂 直樹, 相沢 智康, 新田 勝利, 出村 誠, 河野 敬一, 生物物理, 45, 1, S98, 2005年10月19日
日本生物物理学会, 日本語

1P271 ハロロドプシンCP側チャネル変異体K215Rの光中間体への寄与(光生物 A) 視覚・光受容))
斉藤 裕子, 久保 恵美, 佐藤 真希, 菊川 峰志, 相沢 智康, 加茂 直樹, 新田 勝利, 河野 敬一, 出村 誠, 生物物理, 45, 1, S99, 2005年10月19日
日本生物物理学会, 日本語

Roles of Ser130 and Thr126 in Chloride Binding and Photocycle of pharaonis Halorhodopsin
Sato Maki, Kikukawa Takashi, Araiso Tsunehisa, OKITA Hirotaka, SHIMONO Kazumi, KAMO Naoki, DEMURA Makoto, NITTA Katsutoshi, The journal of biochemistry, 134, 1, 151, 158, 2003年
Japanese Biochemical Society, 英語

創薬研究のための相互作用解析パーフェクト : 低中分子・抗体創薬におけるスクリーニング戦略と実例、in silico解析、一歩進んだ分析技術まで
菊川峰志, 尾瀬農之, 金城政孝, I-7 蛍光偏光測定
羊土社, 2021年12月, 9784758122566, 365p, 日本語, ［分担執筆］

Epigenetics to Optogenetics - A New Paradigm in the Study of Biology　　　　　　　　　　　　　　　
Tamogami, J., Kikukawa, T., Functional Mechanism of Proton Pump-Type Rhodopsins Found in Various Microorganisms as a Potential Effective Tool in Optogenetics
IntechOpen, 2021年06月, ［分担執筆］

Optogenetics: Light-Sensing Proteins and Their Applications in Neuroscience and Beyond　　　　　　　　　　　　　　　
Kikukawa, T, Chapter 4, Functional Mechanism of Cl−-Pump Rhodopsin and Its Conversion into H+ Pump
Springer, 2021年01月, ［分担執筆］

光と生命の事典
菊川峰志, プロテオロドプシン
朝倉書店, 2016年03月, 4254171617, 422, ［分担執筆］

Optogenetics: Light-Sensing Proteins and Their Applications
Kikukawa, T, Kamo, N, Demura, M, Chapter 4, Photochemistry of Halorhodopsin
Springer, 2015年06月, 4431555153, 409, ［分担執筆］

オプトジェネティクス（光遺伝学）～光操作による行動制御技術～　　　　　　　　　　　　　　　
出村誠, 菊川峰志, 加茂直樹, ハロロドプシンの分子構造と機能解析
エヌ・ティー・エス, 2013年, 9784864690713, 日本語, ［分担執筆］

Molecular Photochemistry - Various Aspects　　　　　　　　　　　　　　　
Kikukawa, T, Tamogami, J, Shimono, K, Demura, M, Nara, T, Kamo, N, Chapter 5, Photo-induced Proton Transfers of Microbial Rhodopsins
InTech, 2012年, ［分担執筆］

Retinal conformations in microbial rhodopsins probed by Raman optical activity　　　　　　　　　　　　　　　
Masashi Unno, Tomotsumi Fujisawa, Takashi Kikukawa
19th International Conference on Retinal Proteins, 2022年11月03日, 英語, 口頭発表（招待・特別）
2022年10月30日 - 2022年11月04日, ［招待講演］

A possible gate-opening mechanism of Indibacter alkaliphilus sodium pump rhodopsin　　　　　　　　　　　　　　　
Kikukawa, T.
19th International Conference on Retinal Proteins, 2022年10月31日, 英語, 口頭発表（招待・特別）
2022年10月30日 - 2022年11月04日, ［招待講演］

フラッシュフォトリシス法を用いた微生物ロドプシンの光反応解析　　　　　　　　　　　　　　　
菊川 峰志
日本生物物理学会第58回年会, 2020年09月17日, 英語, シンポジウム・ワークショップパネル（公募）
2020年09月16日 - 2020年09月18日, ［招待講演］

微生物ロドプシンのホモ多量体形成の機能的意義　　　　　　　　　　　　　　　
菊川 峰志
東京大学物性研究所ワークショップ「レチナールタンパク質の光機能発現の物理と化学」, 2019年09月06日, 日本語, 口頭発表（招待・特別）
［招待講演］, ［国内会議］

Cyanobacterial rhodopsin having TSD motif　　　　　　　　　　　　　　　
Kikukawa, T
8th Asia and Oceania Conference on Photobiology, 2017年11月15日, 英語, 口頭発表（招待・特別）
Seoul, Korea, ［招待講演］, ［国際会議］

Light-induced conformational change of inward Cl- pump halorhodopsin　　　　　　　　　　　　　　　
Kikukawa, T, Kamo, N, Demura, M
13th International Conference on Flow Dynamics, 2016年10月11日, 英語, 口頭発表（招待・特別）
［招待講演］, ［国際会議］

時間分解吸収分光で膜蛋白質の反応中間体を捕らえる　　　　　　　　　　　　　　　
菊川 峰志
第10回日本蛋白質科学会年会, 2010年06月, 日本語, シンポジウム・ワークショップパネル（指名）
［招待講演］, ［国内会議］

Important amino acid residues for function of halorhodopsin from Natronomonas pharaonis, a light-driven Cl- pump　　　　　　　　　　　　　　　
Kikukawa, T, Kubo, M, Miyauchi, S, Kamo, N, Demura, M
4th Asia Oceania Conference on Photobiology, 2008年11月, 英語, シンポジウム・ワークショップパネル（指名）
Varanasi, India, ［招待講演］, ［国際会議］

On the sensory rhodopsins in halobacteria, extremely halophilic archaea　　　　　　　　　　　　　　　
Kikukawa, T, Abe, T, Nakao, Y, Kamo, N
15th Anniversary meeting for Korean society of photoscience, 2008年09月, 英語, シンポジウム・ワークショップパネル（指名）
Jeju, Korea, ［招待講演］, ［国際会議］

Analysis of photo-induced proton uptake/release by bacteriorhodopsin and proteorhodopsin using a SnO2 transparent electrode　　　　　　　　　　　　　　　
Tamogami, J, Kikukawa, T, Miyauchi, S, Kamo, N
13th International Conference on Retinal Proteins, 2008年06月, 英語, シンポジウム・ワークショップパネル（指名）
Barcelona, Spain, ［招待講演］, ［国際会議］

Importance of specific hydrogen bonds of archaeal rhodopsins for the binding with transducer protein: what happens when the transducer protein binds to an engineered pharaonis halorhodopsin　　　　　　　　　　　　　　　
Kamo, N, Sudo, Y, Hasegawa, C, Miyauchi, S, Kikukawa, T
12th International Conference on Retinal Proteins, 2006年06月, 英語, シンポジウム・ワークショップパネル（指名）
Awaji, Japan, ［招待講演］, ［国際会議］

生体高分子学実験Ⅰ　　　　　　　　　　　　　　　
北海道大学理学部 生物科学科（高分子機能学）

生物系の分光学　　　　　　　　　　　　　　　
北海道大学理学部 生物科学科（高分子機能学）

生物系の物質輸送論　　　　　　　　　　　　　　　
北海道大学理学部 生物科学科（高分子機能学）

超分子構造科学特論　　　　　　　　　　　　　　　
北海道大学大学院 理学研究院

2021年02月 - 現在
American Society for Biochemistry and Molecular Biology　　　　　　　　　　　　　　　

スギ・ヒノキ由来ペプチドGRPによる花粉－食物アレルギー症候群発症の分子機構解析
科学研究費助成事業
2025年04月 - 2028年03月
相沢 智康, 新井 達也, 久米田 博之, 菊川 峰志, 門間 敬子
日本学術振興会, 基盤研究(B), 北海道大学, 研究分担者, 25K02233

光駆動型重⾦属回収ナノマシンの開発補助事業　　　　　　　　　　　　　　　
2025年度 機械振興補助事業 研究補助
2025年04月 - 2026年03月
公益財団法人 JKA, 研究代表者, 2025M-319

太陽光をエネルギー源とする重金属回収大腸菌の開発　　　　　　　　　　　　　　　
2024年11月 - 2025年10月
公益財団法人 鉄鋼環境基金, 環境研究助成（一般研究）, 研究代表者, C-40-56

過渡現象解析で追求する膜輸送タンパク質の多段階構造変化と輸送素過程の連関
科学研究費助成事業
2022年04月01日 - 2025年03月31日
菊川 峰志, 宮内 正二, 塚本 卓, 海野 雅司
本研究では、膜輸送蛋白質を研究対象とし、多段階の構造変化と、基質の各輸送ステップの関係の解明を目指している。試料としては、イオンポンプとして働く微生物ロドプシンを用いている。この蛋白質は、光で瞬間的に活性化できるため、短寿命で出現する種々の中間体を過渡応答解析によって検出できる。今年度は、Cl-ポンプロドプシンの分子機構について、以下の知見を得た。
1) 細胞質側のCl-輸送に不可欠なアミノ酸残基の同定
イオンポンプロドプシンの細胞質側は疎水的である。H+ポンプでは、この部位に位置する酸性アミノ酸が、高速なH+移動に重要であるが、他のイオンポンプの仕組みは不明であった。そこで、Cl-ポンプにとって重要なアミノ酸を探索したところ、6番目のヘリックスに位置するLeu及びPhe残基の置換によって、透過速度は100倍以上遅くなるが、その近傍に位置するLys残基を同時に置換すると、遅延が大きく回復することを見出した。この結果から、Cl-ポンプでは疎水性アミノ酸だけで輸送経路を構成し、他の親水性成分と相互作用させないことが高速なCl-移動に重要であることが示唆された。
2) Cl-放出・取込み中間体の同定
輸送メカニズムを考察する上で、これらの中間体の同定は必須である。そこで、Cl-選択膜を用いた電気化学的な測定を行なった。基質の放出と取込みは異なるタイミングで起こるため、溶液中の基質濃度の増加と減少を伴う。この濃度変化は非常に微小であり検出は難しいが、本実験では、Cl-ポンプロドプシンをCl-選択膜に吸着させることで、Cl-選択膜近傍で、比較的大きなCl-濃度変化を起こすことを狙った。その結果、濃度変化を膜電位変化として検出することに成功し、さらに、この結果を、Cl-ポンプロドプシンの過渡的な吸収スペクトル変化と比較することで、Cl-放出と取込みが起こる中間体を同定することに成功した。
日本学術振興会, 基盤研究(B), 北海道大学, 23K23843

過渡現象解析で追求する膜輸送タンパク質の多段階構造変化と輸送素過程の連関
科学研究費助成事業 基盤研究(B)
2022年04月01日 - 2025年03月31日
菊川 峰志
日本学術振興会, 基盤研究(B), 北海道大学, 22H02579

アルブミン介在輸送促進の生理的意義：解離促進機構の実証
科学研究費助成事業
2024年04月 - 2025年03月
宮内 正二, 菊川 峰志
日本学術振興会, 基盤研究(C), 東邦大学, 研究分担者, 24K09753

光エネルギーで駆動される物質回収・放出カプセルのボトムアップ構築　　　　　　　　　　　　　　　
科学研究費助成事業 学術変革領域研究(A)
2022年06月16日 - 2024年03月31日
菊川 峰志
日本学術振興会, 学術変革領域研究(A), 北海道大学, 22H05389

同生物種がもつ正と負の２つの光センサ分子の機能変換から機能の違いを生む要因を探る
科学研究費助成事業
2021年04月01日 - 2024年03月31日
田母神 淳, 菊川 峰志
高度好塩菌に存在する２つのセンサリーロドプシン（SRI、SRII）は互いに異なる走光性に関与するが、その機能の違いを生む要因が何かを明らかにすることが本研究の目的である。これを達成するために、本研究では同生物種（Haloarcula vallismortis）由来でかつ安定性に優れたHvSRIとHvSRIIを用いた物性解析と機能変換実験を行った。レチナール結合部位近傍のアミノ酸残基に着目した部位特異的変異導入実験から、HvSRIIのN83残基をHvSRIと同じLeu残基に置換したとき、吸収波長と光反応がHvSRIに最も近づき、2つのSR間の違いを決める重要残基の1つであることが明らかになった。
日本学術振興会, 基盤研究(C), 松山大学, 21K06091

アルブミン介在肝取り込み促進機構の解明とその促進機構モデルの新展開
科学研究費助成事業 基盤研究(C)
2021年04月01日 - 2024年03月31日
宮内 正二, 菊川 峰志
基礎学問から応用学問まで薬学領域において幅広く受け入れられている概念”free drug theory (FDT)”に従わない、アルブミン介在取り込み促進機構解明を詳細に解析するための最適な実験系の構築を行った。先ず、アフリカツメガエル卵母細胞発現系を用いて、アルブミン結合率の高い薬物を輸送する有機アニオン輸送担体（OATP,OAT）の輸送活性を示す発現系を確立した。構築した輸送担体発現系は高い輸送活性を示し、アルブミン存在下でも詳細な速度論的な解析が可能なものである。これら発現系はイオン駆動力が解明されていない有機アニオン輸送担体の分子輸送機構の解明、エネルギー共役系の解明にも応用できる有用な実験系であることが明らかになった。実際に、アルブミン存在下で輸送促進機構、特に、アルブミンの輸送駆動力に及ぼす影響について検討を進めている。更に、これまでアルブミン促進機構が報告されている臓器の抽出RNAを用いて有機アニオン輸送担体の単離、輸送実験系を確立した。これにより脳毛細血管や腎毛細血管において報告されているアルブミン促進機構の詳細なメカニズムとその生理的意義を今後検討する。
一方、並行して進めている蛍光物質利用した蛍光相関分光分析法（Fluorescence Correlation Spectroscopy：FCS）によりアルブミン介在肝取り込み促進を詳細に解析出来る実験系の構築を行っている。今年度明らかにした知見として、蛍光強度の自己相関関数の測定には、蛍光物質の励起状態安定性が必須であること、アルブミン促進を示す蛍光物質はFCSには適切で無いことである。今後、FCS解析に適切な蛍光物質の検討を現在行っている。
日本学術振興会, 基盤研究(C), 東邦大学, 21K06519

膜輸送タンパク質の光エネルギー共役を基盤とする環境浄化大腸菌の開発　　　　　　　　　　　　　　　
環境研究助成
2021年11月 - 2022年11月
菊川 峰志
公益財団法人 住友財団, 研究代表者, 競争的資金

毒物耐性スクリーニングを用いた膜輸送タンパク質の革新的機能改変　　　　　　　　　　　　　　　
一般助成
2021年04月 - 2022年03月
菊川 峰志
公益財団法人 秋山記念生命科学振興財団, 研究代表者, 競争的資金

多彩な基質輸送能力を有する輸送担体を制御する輸送分子機構解明
科学研究費補助金（基盤研究(C)）
2018年04月 - 2021年03月
宮内 正二
文部科学省, 競争的資金

光駆動型ナトリウムポンプ：イオン輸送素過程の解明と機能改変の試み
科学研究費補助金(基盤研究(C))
2017年 - 2019年
菊川 峰志
文部科学省, 研究代表者, 競争的資金

トランスポーターの基質輸送サイクルを決定する分子弁の同定と機能解析
科学研究費補助金(基盤研究(C))
2014年 - 2016年
宮内 正二
本研究では、トランスポーターの基質輸送サイクルを決定する分子弁の同定とその輸送分子機構を明らかにすることを目的として、様々な起電性輸送担体をモデル輸送担体として用い、以下に示す研究成果を得た。
(1) ヒトH+/オリゴペプチド共輸送担体 (hPEPT1)の基質輸送の方向性を決定する分子弁の実体を明らかにした。
(2) Na+/モノカルボン酸共輸送担体 (hSMCT1) の方向性を決定する分子弁の存在を明らかにした。
文部科学省, 基盤研究(C), 東邦大学, 競争的資金, 26460044

光駆動型ナトリウムポンプの生理的意義とイオン輸送機構の解明
科学研究費補助金(基盤研究(C))
2014年 - 2016年
菊川 峰志
微生物型ロドプシンは、色素レチナールを内包する膜タンパク質である。レチナールの光吸収をきっかけとして、複数の構造の異なる中間体を経由する光反応サイクルを周り、この間にイオンの膜輸送や光情報伝達を行う。本研究では、外向きにNa+を輸送するNa+ポンプ型ロドプシン（NaR）の機能解析を行い、以下の成果を得た。１）タンパク質表面へのカチオン結合が暗状態の構造を変調し、光反応初期に出現する中間体を決定する。２）NaRのNa+取込みと放出が、O中間体の生成・崩壊に同期して起こることを、Na+濃度変化の直接観測から明らかとした。
文部科学省, 基盤研究(C), 北海道大学, 研究代表者, 競争的資金, 26440042

イオン輸送を伴うトランスポーターの輸送分子機構の解明
科学研究費補助金(基盤研究(C))
2011年 - 2013年
宮内 正二, 菊川 峰志
H+ 濃度勾配を利用してジペプチドから医薬品まで能動輸送するH+/オリゴペプチド輸送担体 (PEPT) の輸送分子機構の解明をおこなった．PEPT は，第二膜貫通領域 (TDM2) に存在する輸送活性中心である His57 のプロトン化により活性調節されているが，どの様なメカニズムで活性調節をおこなっているか不明である．His57のプロトン化による輸送活性の制御がどの様な分子メカニズムによるかを検討するために，PEPTsoの結晶構造（バクテリアにおけるPEPT類似輸送担体）を基にHis57と相互作用する可能性が高いSer302の変異体を作成し，輸送活性のpH依存性を検討した．結晶構造情報によれば，His57とSer302との距離は水素結合が可能な2.7Åにある． Ser302をAla, Thr, Valに変化させた変異体をアフリカツメガエル卵母細胞に発現させGly-Sar (典型的なPEPTの基質)の取り込み輸送活性のpH依存性を検討した． Ala, Thrの変異体は野生型hPEPT1に比べて輸送活性は低下したが，輸送活性は保持していた．取り込み輸送活性のpH依存性は，Ala, Thrの変異体について，ベル型の至適pHがアルカリ側にシフトした．S302V変異体についてはHis57が水素結合を形成することが出来ない状態にあり，輸送活性が見られなかった．様々な代謝酵素において，活性中心のSerの水酸基がHisのイミダゾ-ル基と水素結合を形成し，His残基側鎖のプロトン化に伴う水素結合ネットワーク変化により，活性調節がなされている．Gly-Sar輸送活性のpH依存性のアルカリ側へのシフトは，hPEPT1にもよく似たpH活性調節機構が備わっておりHis57とSer302の水素結合が活性調節に重要であるという可能性を示唆したものである．
文部科学省, 基盤研究(C), 松山大学->東邦大学, 連携研究者, 競争的資金, 23590062

光駆動型イオンポンプ：ハロロドプシンのクロライド輸送素過程の解明
科学研究費補助金(基盤研究(C))
2011年 - 2013年
菊川 峰志, 宮内 正二
高度好塩菌の細胞膜に存在するハロロドプシン（HR)は、光エネルギーを用いて、Cl-を細胞外から細胞内へ輸送する蛋白質である。光で活性化できるという利点に加え、輸送活性などの測定手法の確立によって、HRは輸送分子機構を詳細に解析できる優れたモデル蛋白質となってきている。本研究では、輸送に伴う分子内イベントの解析と、そこに係わる重要アミノ酸残基の同定を通して、HRの分子機構を考察する。今年度は、以下の二つの成果を得た。１．HRが光反応サイクル中に発生する起電力の時間分解測定：誘電性薄膜であるコロジオン膜にHRを吸着させ、この膜を用いた電気化学セルを構築した。誘電性薄膜はコンデンサ、HRは電池とみなせるため、Cl-輸送に伴う輸送起電力は、光誘導電流として観測される。電気化学セルにナノ秒パルスレーザを照射したところ、数ミリ秒で最大となる誘導電流を観測した。これは、光反応サイクルにおけるO中間体の生成・崩壊のタイミングとほぼ一致しており、この中間体の前後で、Cl-の長距離の移動が起こることが示唆された。２．H.salinarum由来のHR（HsHR）の大腸菌発現系を用いた調製法の確立：HRの研究には、HsHRとN.pharaonis由来のHR（NpHR）が主に用いられてきた。両者には、輸送基質の種類や光反応サイクルなど、興味深い違いがあることが知られており、これらを比較することで、Cl-輸送機構をより詳細に検討できるものと期待される。NpHRは、大腸菌における機能的大量発現系が確立しているが、HsHRでは、これがなかった。そこで、HsHRの大腸菌発現系を用いた調製を試みた。その結果、HsHRは、不活性な状態で大腸菌膜に発現するが、可溶化時にレチナールを添加することで、活性型のHsHRを得られることが明らかとなった。この手法によって、HsHR変異体を容易に調製できるようになった。
文部科学省, 基盤研究(C), 北海道大学, 研究代表者, 競争的資金, 23510251

トランスポーターの輸送分子機構解明の新展開:光駆動性ポンプをモデルタンパクとして研究
科学研究費補助金(基盤研究(C))
2008年 - 2010年
宮内 正二, 菊川 峰志
光駆動性クロライドポンプの輸送分子機構を明らかにすることを研究目的として研究を行い,輸送活性を様々な物理化学的手法より測定し,以下の結果を得た.(1)種々の変異体を作成し,その変異体におけるCl-結合能を測定した.結合に対して重要な残基を特定した.Arg123,Ser130,Asp252である.(2)Cl^-の細胞外から細胞内へのタンパク内移動には,Ser130およびAsp252が重要であることが明らかとなった.(3)Ser130は,Cl^-が細胞質側から細胞外側に逆流が起こらないようにする分子弁として機能していることが明らかとなった
文部科学省, 基盤研究(C), 北海道大学->松山大学, 連携研究者, 競争的資金, 20590031

光駆動型アニオンポンプ蛋白質:ハロロドプシンの分子機構
科学研究費補助金(基盤研究(C))
2007年 - 2009年
菊川 峰志, 宮内 正二
高度好塩菌の細胞膜に存在するハロロドプシンは、光エネルギーを用いて、Cl-イオンを細胞外から細胞内へ輸送するイオンポンプ膜タンパク質である。その分子機構解明に寄与するため、野生型、及び、アミノ酸置換した複数の変異体を用いた解析を行い、以下の知見を得た。(1)活性中心近傍の酸性及び芳香族アミノ酸残基は、輸送の初期過程において重要な役割を果たす。(2)輸送サイクル中に現れる、Cl-濃度依存的な二つの中間体の平衡状態は、Cl-放出過程を反映している。(3)Cl-放出時には、蛋白質の細胞質側チャネルが水和し、Cl-放出状態が安定化する。
文部科学省, 基盤研究(C), 北海道大学, 研究代表者, 競争的資金, 19614001

ハロロドプシンのクロライド輸送機構解明と3量体形成の意義
科学研究費補助金(基盤研究(B))
2007年 - 2009年
加茂 直樹, 宮内 正二, 鷲見 正人, 菊川 峰志, 奈良 敏文, 宮内 正二
ハロロドプシン(HR)は高度好塩菌の膜に発現している光エネルギーで駆動されるClイオンポンプである。Xenopusオーサイトに発現させ、光照射による膜電流の測定より、活性を測定する新しい方法を開発した。非常に定量性優れていた。大腸菌の発現系を用い、種々のHRの変異体を作成し、活性と光化学反応を調べた。Arg123が輸送に重要であることを明らかにした。NからO中間体への変化の際に、ClイオンがHR内部から細胞内部へ放出され、O→HR'の過程でClが細胞外からHRへ取り込まれることを明らかにした。HRは界面活性剤中でも3量体を形成していることを明らかにした。3量体はモノマーより安定であった。
文部科学省, 基盤研究(B), 北海道大学->松山大学, 連携研究者, 競争的資金, 19390009

アフリカツメガエル卵母細胞を用いた電位発生的輸送担体研究の新しい展開
科学研究費補助金(基盤研究(C))
2006年 - 2007年
宮内 正二, 菊川 峰志
1.光駆動性クロライドポンプ(HR)のクロライド輸送機構の解明光駆動性クロライドポンプ(HR)のクロライド輸送機構を電気生理学的に精密に検討することが出来る系を確立した.世界ではじめてこの実験に成功している.この実験の他では出来ないユニークな点は,膜電位をかけながら,Cl^-の輸送が実験出来ることである.これを利用して,HR1分子当たりの「輸送能力」と1秒間に輸送するイオン輸送能力を見積もることを可能とした.これで,様々な変異体の輸送活性を精度良く見積もることができるようになった.輸送における輸送中間体の役割を明らかにするために最も重要な事柄のひとつである.2.オリゴペプチド輸送担体(hPEPT1)の基質輸送機構の解明hPEPT1がトランスポーターでありながら,特殊な基質存在下では,基質認識部位における構造変化によりH^+チャンネルとしても働き得ること見いだした.この知見により、トランスポーターは(チャネル)+(基質スイッチ)の2つの機構からなり,これらを協調的に機能させ,基質を輸送するという作業仮説に至った.この性質は,上紀のCl_-の膜透過過程にも含まれ,膜透過の本質であることも明らかになった.3.ヒトNa^+/モノカルボン酸共輸送体(hSMCT)の基質認識機構の解明構造の異なるモノカルボン酸を用いてhSMCTの斯質認識に関する法則を明らかにした.化合物が長いほど親和性は...
文部科学省, 基盤研究(C), 北海道大学, 連携研究者, 競争的資金, 18590028

抗菌薬排出蛋白質の分子機構-細菌の小型排出蛋白質を例として-
科学研究費補助金(若手研究(B))
2004年 - 2006年
菊川 峰志
ほぼ全ての生物種の細胞膜には,細胞にとって都合の悪い様々な毒物を認識し,能動的に細胞外へと排出する多剤排出蛋白質が存在している.これらの蛋白質は,感染症治療の大きな障害である多剤耐性菌株の出現と密接に係わっている他,分子科学の面からも,構造上相関の無い多様な薬物の認識/排出という興味深い機能を実現している.本研究では,多剤排出蛋白質の中で,最もサイズが小さいSMR(small multidrug resistance)ファミリーに属する蛋白質の分子機構解明を目指した研究を行ってきた.様々な測定手法を適用するためには,蛋白質の高純度な精製が必要なため,SMR蛋白質の大腸菌における大量発現・精製を重要な課題と位置付けて研究を行った.融合タグを用いる方法で,大腸菌のEmrEの大量発現と融合蛋白質の精製は実現できたが,残念ながら,融合タグの分離が依然として困難であった.有機溶媒に溶けるほどのSMRの脂溶性の高さが,プロテアーゼ処理とその後の蛋白質の回収を困難にしていると考えられる.一方,これと平行して,枯草菌のEbrABの膜内トポロジーの決定と,その制御機構を明らかとする研究を行い,興味深い知見を得るに至った.これまでのSMRの研究は,主にEmrEを用いて行われてきた.膜内ではホモダイマーとして機能すると考えられているが,その膜内トポロジーについては,互いに異なる報告がされてきている...
文部科学省, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 16790029

膜蛋白複合体における情報伝達機構:高度好塩菌の光受容を例として
科学研究費補助金(基盤研究(B))
2004年 - 2006年
加茂 直樹, 奈良 敏文, 宮内 正二, 菊川 峰志, 鷲見 正人
生体膜を介する諸機能において,蛋白質複合体で機能する例は多くある.その1つの例として,本研究では高度好塩菌の走光性の受容体(フォボロドプシン,または,センソリーロドプシン)および受容体とトランスジューサ蛋白との相互作用の研究を行った.受容体とトランスジューサは共に膜蛋白である.得られた結果は次の通りである.1.微生物ロドプシンでは,シッフ塩基の近傍に存在するアルギニンは完全に保存されている.このアルギニンの変異体を作成し,この残基の役割を検討した.2.容体が光を受けると,Fヘリックスのコンフォメーションが変化することを示した.3.FTIRの結果から,受容体分子の中の水分子の水素結合が変化することを示した.4.固体NMRの手法を用いて,トランスジューサのダイナミクス変化が,受容体であるフォボロドプシンと結合すると変化することを示した.5.酸化スズの透明電極を用いて,フォボロドプシンの光照射に伴うプロトンの移動を調べ,Asp193からプロトンが放出される事およびこの残基のpKaがトランスジューサとの結合によって変化することを示した.6.トランスジューサの細胞質内の可溶性部分が受容体であるフォボロドプシンと相互作用していることをしました.7.両膜蛋白は膜内で3本の水素結合で相互作用していることを,変異体の結合実験から示した.また,両蛋白のvan deer Waals相互作用も結合に...
文部科学省, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 16390012

アラメシチンのチャネル形成過程-膜脂質の動的構造変化の解析から-
科学研究費補助金(奨励研究(A))
1998年 - 1999年
菊川 峰志
アラメシチンは生体膜に取り付き、膜に電位差が加わると数分子が会合したチャネルを形成する抗生ペプチドである。チャネルの開状態モデル-Barrel Stave Model-は広く受け入られている。しかし、このペプチドがチャネル形成前(閉状態)に膜のどの部分(深さ方向)にどのような構造をとって取り付いているのか、また、どのようなメカニズムで膜を貫通するようになるのかという問題は未だ解決されていない。この問題の解明に、「アラメシチンと脂質との相互作用」という新しい視点に立ち、昨年度から、主に蛍光性脂質を導入したリポソームを用いて取り組んできた。今年度は、液晶相での脂質の頭部及び鎖部の運動性が、アラメシチンにより修飾される様子の詳細な検討を試みた。得られた知見をまとめると以下のようになる。1.脂質運動性の修飾はあるアラメシチン濃度を境に急峻になる。アラメシチンのcooperativityが反映していると考えられる。アラメシチンの侵入が脂質の相構造に大きな擾乱となっていることがわかる。2.脂質組成によって脂質の頭部及び鎖部の運動が異なる修飾を受ける。これは、アラメシチンの存在位置がアラメシチン濃度とともに変化していることを示している。3.脂質運動性の修飾は、微小量のアラメシチン濃度(脂質の3/100程度)で見られる。したがって、アラメシチンは近傍の脂質分子とだけ相互作用するわけではなく、...
文部科学省, 奨励研究(A), 北海道大学, 研究代表者, 競争的資金, 10771265