Aug. 2025, Communications Biology, Reviewer of the Month　　　　　　　　　　　　　　　

Oct. 2005, Hokkaido University, Nitobe Prize　　　　　　　　　　　　　　　
TSUKAMOTO Takashi

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, 02 Oct. 2025, [Peer-reviewed], [International Magazine]
English, Scientific journal, Microbial rhodopsins are photoactive membrane proteins known for transporting small inorganic ions such as H+, Cl-, and Na+. Their compact structure─comprising seven transmembrane helices─has long been thought to limit their substrate range to such ions. Here, we report that several anion-pumping rhodopsins can also transport organic anions. In particular, a rhodopsin from cyanobacteria transports bulky organic anions, including those with a benzene ring, with volumes up to ∼120 Å3─five times larger than Cl-. These anions bind in the dark state and are translocated upon photoactivation, via a mechanism similar to Cl-. Notably, only anions with pKa values below 2 are transported, suggesting that negative charge is essential for binding. This study provides the first evidence that naturally occurring proteins can use light to transport organic compounds across membranes. These findings broaden the functional scope of microbial rhodopsins and open new possibilities for light-driven transport of organic ions.

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, 301, 10, 110703, 110703, 11 Sep. 2025, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Red cell shape regulation by band 3–ankyrin–spectrin linkage: implications for clinical severity of bovine hereditary spherocytosis
Kosuke Miyazono, Mizuki Tomihari, Nobuto Arashiki, Ichiro Koshino, Yayoi Otsuka-Yamasaki, Kota Kizaki, Takashi Tsukamoto, Osamu Inanami, Ayumi Deguchi, Eri Kitaguchi, Makoto Demura, Narla Mohandas, Yuichi Takakuwa, Mutsumi Inaba
Blood Red Cells & Iron, 1, 1, 100003, Jun. 2025, [Peer-reviewed]
Scientific journal

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, 2257, 2265, 17 Sep. 2024, [Peer-reviewed], [Last author, Corresponding author]
Scientific journal

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, Sep. 2024, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

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, 25 Oct. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, 29 Sep. 2023, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, 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.

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, 18 May 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Concerted primary proton transfer reactions in a thermophilic rhodopsin studied by time-resolved infrared spectroscopy at high temperature.
Kunisato Kuroi, Takashi Tsukamoto, Naoya Honda, Yuki Sudo, Yuji Furutani
Biochimica et biophysica acta. Bioenergetics, 1864, 3, 148980, 148980, 18 Apr. 2023, [Peer-reviewed], [Lead author], [International Magazine]
English, Scientific journal, The primary proton transfer reactions of thermophilic rhodopsin, which was first discovered in an extreme thermophile, Thermus thermophilus JL-18, were investigated using time-resolved Fourier transform infrared spectroscopy at various temperatures ranging from 298 to 343 K (25 to 70 °C) and proton transport activity analysis. The analyses were performed using counterion (D95E, D95N, D229E, and D229N) and proton donor mutants (E106D and E106Q) as well. First, the initial proton transfer from the protonated retinal Schiff base (PRSB) to D95 was identified. The temperature dependency showed that the proton transfer reaction in the intermediate states dramatically changed above 318 K (45 °C). In addition, the proton transfer reaction correlated well with the structural change from turn to β-strand in the protein moiety, suggesting that this step may be regulated by the rigidity of the loop region. We also elucidated that the proton transfer reaction from proton donor E106 to the retinal Schiff base occurred synchronously with the primary proton transfer from the PRSB to D95. Surprisingly, we discovered that the direction of proton transfer was regulated by the secondary counterion, D229. Comparative analysis of Gloeobacter rhodopsin from the mesophile, Gloeobacter violaceus, highlighted that the primary proton transfer reactions in thermophilic rhodopsin were optimized at high temperatures partly due to the specific turn to β-strand structural change. This was not observed in Gloeobacter rhodopsin and other related proteins such as bacteriorhodopsin.

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, 30 Sep. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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, American Chemical Society (ACS), 13 Sep. 2022, [Peer-reviewed]
Scientific journal

Real-Time Identification of Two Substrate-Binding Intermediates for the Light-Driven Sodium Pump Rhodopsin.
Tomoya Kato, Takashi Tsukamoto, Makoto Demura, Takashi Kikukawa
The Journal of biological chemistry, 100792, 100792, 18 May 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

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, 12 Apr. 2021, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, 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.

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, 06 Sep. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids.
Ayumi Yamamoto, Takashi Tsukamoto, Kenshiro Suzuki, Eri Hashimoto, Yoshihiro Kobashigawa, Kousuke Shibasaki, Takeshi Uchida, Fuyuhiko Inagaki, Makoto Demura, Koichiro Ishimori
Biophysical journal, 118, 11, 2853, 2865, 29 Apr. 2020, [Peer-reviewed], [Lead author], [International Magazine]
English, Scientific journal, We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.

Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained.
Satoshi Yasuda, Tomoki Akiyama, Sayaka Nemoto, Tomohiko Hayashi, Tetsuya Ueta, Keiichi Kojima, Takashi Tsukamoto, Satoru Nagatoishi, Kouhei Tsumoto, Yuki Sudo, Masahiro Kinoshita, Takeshi Murata
Journal of chemical information and modeling, 60, 3, 1709, 1716, 23 Mar. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, We develop a new methodology best suited to the identification of thermostabilizing mutations for an intrinsically stable membrane protein. The recently discovered thermophilic rhodopsin, whose apparent midpoint temperature of thermal denaturation Tm is measured to be ∼91.8 °C, is chosen as a paradigmatic target. In the methodology, we first regard the residues whose side chains are missing in the crystal structure of the wild type (WT) as the "residues with disordered side chains," which make no significant contributions to the stability, unlike the other essential residues. We then undertake mutating each of the residues with disordered side chains to another residue except Ala and Pro, and the resultant mutant structure is constructed by modifying only the local structure around the mutated residue. This construction is based on the postulation that the structure formed by the other essential residues, which is nearly optimized in such a highly stable protein, should not be modified. The stability changes arising from the mutations are then evaluated using our physics-based free-energy function (FEF). We choose the mutations for which the FEF is much lower than for the WT and test them by experiments. We successfully find three mutants that are significantly more stable than the WT. A double mutant whose Tm reaches ∼100 °C is also discovered.

Functional importance of the oligomer formation of the cyanobacterial H+ pump Gloeobacter rhodopsin
Azusa Iizuka, Kousuke Kajimoto, Tomotsumi Fujisawa, Takashi Tsukamoto, Tomoyasu Aizawa, Naoki Kamo, Kwang-Hwan Jung, Masashi Unno, Makoto Demura, Takashi Kikukawa
Scientific Reports, 9, 1, Dec. 2019, [Peer-reviewed]
English, Scientific journal

Quantitation of the neural silencing activity of anion channelrhodopsins in Caenorhabditis elegans and their applicability for long-term illumination
Taro Yamanashi, Misayo Maki, Keiichi Kojima, Atsushi Shibukawa, Takashi Tsukamoto, Srikanta Chowdhury, Akihiro Yamanaka, Shin Takagi, Yuki Sudo
Scientific Reports, 9, 1, Springer Science and Business Media LLC, Mar. 2019, [Peer-reviewed]
Scientific journal

Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl− concentrations
Takashi Tsukamoto, Chihiro Kikuchi, Hiromu Suzuki, Tomoyasu Aizawa, Takashi Kikukawa, Makoto Demura
Scientific Reports, 8, 1, 13445, 13445, Springer Science and Business Media LLC, Dec. 2018, [Lead author, Corresponding author], [International Magazine]
English, Scientific journal, 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-.

Correction to “High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment”
Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, Yasuhisa Mizutani
The Journal of Physical Chemistry B, 122, 42, 9826, 9826, American Chemical Society ({ACS}), 25 Oct. 2018, [Peer-reviewed]
Scientific journal

High Thermal Stability of Oligomeric Assemblies of Thermophilic Rhodopsin in a Lipid Environment
Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, Yasuhisa Mizutani
The Journal of Physical Chemistry B, 122, 27, 6945, 6953, American Chemical Society ({ACS}), Jul. 2018, [Peer-reviewed]
Scientific journal

Production of a Light-Gated Proton Channel by Replacing the Retinal Chromophore with Its Synthetic Vinylene Derivative
Riho Takayama, Akimasa Kaneko, Takashi Okitsu, Satoshi P. Tsunoda, Kazumi Shimono, Misao Mizuno, Keiichi Kojima, Takashi Tsukamoto, Hideki Kandori, Yasuhisa Mizutani, Akimori Wada, Yuki Sudo
Journal of Physical Chemistry Letters, 9, 11, 2857, 2862, American Chemical Society, 07 Jun. 2018, [Peer-reviewed]
English, Scientific journal

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, 29 Mar. 2018, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, 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.

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, Royal Society of Chemistry ({RSC}), 31 Jan. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Comparative evaluation of the stability of seven-transmembrane microbial rhodopsins to various physicochemical stimuli
Naoya Honda, Takashi Tsukamoto, Yuki Sudo
CHEMICAL PHYSICS LETTERS, 682, 6, 14, Aug. 2017, [Peer-reviewed]
English, Scientific journal

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, Mar. 2017, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Demonstration of a Light-Driven SO42- Transporter and Its Spectroscopic Characteristics
Akiko Niho, Susumu Yoshizawa, Takashi Tsukamoto, Marie Kurihara, Shinya Tahara, Yu Nakajima, Misao Mizuno, Hikaru Kuramochi, Tahei Tahara, Yasuhisa Mizutani, Yuki Sudo
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 139, 12, 4376, 4389, Mar. 2017, [Peer-reviewed], [Lead author]
English, Scientific journal

A phylogenetically distinctive and extremely heat stable light-driven proton pump from the eubacterium Rubrobacter xylanophilus DSM 9941(T)
Kanae Kanehara, Susumu Yoshizawa, Takashi Tsukamoto, Yuki Sudo
SCIENTIFIC REPORTS, 7, 44427, Mar. 2017, [Peer-reviewed]
English, Scientific journal

An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli
Satoko Doi, Takashi Tsukamoto, Susumu Yoshizawa, Yuki Sudo
SCIENTIFIC REPORTS, 7, 41879, Feb. 2017, [Peer-reviewed]
English, Scientific journal

Live-cell single-molecule imaging of the cytokine receptor MPL for analysis of dynamic dimerization
Akihiko Sakamoto, Takashi Tsukamoto, Yuji Furutani, Yuki Sudo, Kazuyuki Shimada, Akihiro Tomita, Hitoshi Kiyoi, Takashi Kato, Takashi Funatsu
Journal of Molecular Cell Biology, 8, 6, 553, 555, Dec. 2016, [Peer-reviewed]
English

X-ray Crystallographic Structure of Thermophilic Rhodopsin IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION
Takashi Tsukamoto, Kenji Mizutani, Taisuke Hasegawa, Megumi Takahashi, Naoya Honda, Naoki Hashimoto, Kazumi Shimono, Keitaro Yamashita, Masaki Yamamoto, Seiji Miyauchi, Shin Takagi, Shigehiko Hayashi, Takeshi Murata, Yuki Sudo
JOURNAL OF BIOLOGICAL CHEMISTRY, 291, 23, 12223, 12232, Jun. 2016, [Peer-reviewed], [Lead author]
English, Scientific journal

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 (BBA) - Bioenergetics, 1847, 8, 748, 758, Elsevier {BV}, Aug. 2015, [Peer-reviewed]
Scientific journal

Converting a Light-Driven Proton Pump into a Light-Gated Proton Channel
Keiichi Inoue, Takashi Tsukamoto, Kazumi Shimono, Yuto Suzuki, Seiji Miyauchi, Shigehiko Hayashi, Hideki Kandori, Yuki Sudo
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 137, 9, 3291, 3299, Mar. 2015, [Peer-reviewed]
English, Scientific journal

Structural and functional roles of the N- and C-terminal extended modules in channelrhodopsin-1
Satoko Doi, Arisa Mori, Takashi Tsukamoto, Louisa Reissig, Kunio Ihara, Yuki Sudo
PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 14, 9, 1628, 1636, 2015, [Peer-reviewed]
English, Scientific journal

Irreversible Trimer to Monomer Transition of Thermophilic Rhodopsin upon Thermal Stimulation
Takashi Tsukamoto, Makoto Demura, Yuki Sudo
JOURNAL OF PHYSICAL CHEMISTRY B, 118, 43, 12383, 12394, Oct. 2014, [Peer-reviewed], [Lead author]
English, Scientific journal

Molecular and evolutionary aspects of microbial sensory rhodopsins.
Inoue K, Tsukamoto T, Sudo Y
Biochimica et biophysica acta, 1837, 5, 562, 577, May 2014, [Peer-reviewed]
Scientific journal

Sensory Rhodopsins
Jan. 2014, [Peer-reviewed]

Role of S-Palmitoylation on IFITM5 for the Interaction with FKBP11 in Osteoblast Cells
Takashi Tsukamoto, Xianglan Li, Hiromi Morita, Takashi Minowa, Tomoyasu Aizawa, Nobutaka Hanagata, Makoto Demura
PLOS ONE, 8, 9, e75831, Sep. 2013, [Peer-reviewed], [Lead author, Corresponding author]
English, Scientific journal

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile
Takashi Tsukamoto, Keiichi Inoue, Hideki Kandori, Yuki Sudo
JOURNAL OF BIOLOGICAL CHEMISTRY, 288, 30, 21581, 21592, Jul. 2013, [Peer-reviewed], [Lead author]
English, Scientific journal

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, 14 Feb. 2013, [Peer-reviewed], [Lead author]
English, Scientific journal

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, Jun. 2012, [Peer-reviewed], [Lead author]
English, Scientific journal

Expression of salinarum halorhodopsin in Escherichia coli cells: Solubilization in the presence of retinal yields the natural state
Yasutaka Yamashita, Takashi Kikukawa, Takashi Tsukamoto, Masakatsu Kamiya, Tomoyasu Aizawa, Keiichi Kawano, Seiji Miyauchi, Naoki Kamo, Makoto Demura
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1808, 12, 2905, 2912, Elsevier {BV}, Dec. 2011, [Peer-reviewed]
Scientific journal

光で働く微生物のロドプシン-どのように?なんのために?を紐解いてきた50年
塚本卓, 出村誠, 化学, 76, 4, 2021, [Invited], [Lead author]

ヒト便試料の保存条件とメタボローム変動のNMR解析(Impact of sample storage conditions on NMR-based human fecal metabolomics)
宋 子豪, 包 克非, 北田 直也, 清水 由宇, 菊池 摩仁, 熊木 康裕, 大西 裕季, 塚本 卓, 菊川 峰志, 出村 誠, 中村 公則, 綾部 時芳, 山村 凌大, 中村 幸志, 玉腰 暁子, 相沢 智康, 腸内細菌学雑誌, 34, 2, 148, 148, Apr. 2020
(公財)腸内細菌学会, English

Overexpression of stable isotope-labeled cecropin P1 by using calmodulin-fusion protein system and NMR researches
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

微生物型ロドプシンTRのX線結晶構造解析
水谷健二, 橋本直記, 塚本卓, 須藤雄気, 村田武士, KEK Progress Report (Web), 2016-8, ROMBUNNO.242 (WEB ONLY), Jan. 2017
Japanese

Identification of thermostabilizing mutations for the Thermophilic Rhodopsin based on statistical thermodynamics　　　　　　　　　　　　　　　
Sayaka Nemoto, Satoshi Yasuda, Takashi Tsukamoto, Yuki Sudo, Masahiro Kinoshita, Takeshi Murata, 第54回 日本生物物理学会年会 つくば国際会議場、2016年11月25-27日, Nov. 2016

Retinal Proteins in Thermophilic Bacteria
Takashi TSUKAMOTO, Yuki SUDO, Seibutsu Butsuri, 55, 2, 092, 094, 2015
Biophysical Society of Japan, Japanese

時間分解赤外分光計測による好熱性ロドプシンTRの光反応解析
黒井邦巧, 塚本卓, 本田尚也, 須藤雄気, 古谷祐詞, 分子科学討論会講演プログラム&要旨(Web), 9th, ROMBUNNO.4P066 (WEB ONLY), 2015
Japanese

Molecular and evolutionary aspects of microbial sensory rhodopsins
Keiichi Inoue, Takashi Tsukamoto, Yuki Sudo, BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS, 1837, 5, 562, 577, May 2014
English, Book review

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, 31 Aug. 2012
Japanese

ナノディスクを用いた膜タンパク質ハロロドプシンの機能解析
山本愛弓, 塚本卓, 小橋川敬博, 内田毅, 出村誠, 稲垣冬彦, 石森浩一郎, 日本蛋白質科学会年会プログラム・要旨集, 12th, 63, 31 May 2012
Japanese

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, 31 May 2012
Japanese

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, 27 Apr. 2012
Japanese

膜タンパク質Ifitm5の立体構造研究;溶液NMR法による立体構造解析に向けた試料調製
塚本卓, LI Xiang Lan, 花方信孝, 出村誠, Abstr Annu Meet NMR Soc Jpn, 48th, 166, 167, 10 Nov. 2009
Japanese

1P091 Dissociation of Halorhodopsin Trimer in Detergent System(Membrane proteins,Oral Presentations)
Tsukamoto Takashi, Sasaki Takanori, Kubo Megumi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Demura Makoto, Biophysics, 47, 1, S46, 20 Nov. 2007
The Biophysical Society of Japan General Incorporated Association, English

Unexpected Insights Gained from Experimental Studies of Anion Channelrhodopsins
Takashi Tsukamoto
第63回日本生物物理学会年会, 24 Sep. 2025, Invited oral presentation
24 Sep. 2025 - 26 Sep. 2025, 40198229, [Invited]

アニオンチャネルロドプシンの２段階のゲート閉鎖過程における過渡的な分子内イベントの解析
塚本卓
第51回生体分子科学討論会, 27 Jun. 2025, Oral presentation
26 Jun. 2025 - 27 Jun. 2025, 42185177

Anion Concentration Dependency on the Photocycle of PsuACR1: Implications for the Impairment of its Fast Channel Function.　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
18th International Conference on Retinal Proteins, 24 Sep. 2018, English, Poster presentation
[International presentation]

Substrate anion concentration significantly affects the fast channel function of Proteomonas sulcata anion channelrhodopsin-1　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第56回年会, 15 Sep. 2018, English, Oral presentation
[Domestic Conference]

Photochemical Properties of Microbial Rhodopsin Pumps and Channels　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
APSBMS 2018 Annual Meeting, 21 Jul. 2018, English, Invited oral presentation
[Invited], [International presentation]

塩濃度に依存したアニオンチャネルロドプシンの光化学的性質の変化　　　　　　　　　　　　　　　
塚本 卓
日本生物物理学会北海道支部例会, 17 Mar. 2018, Japanese, Oral presentation
[Domestic Conference]

Light-driven Cl- transport mechanism of Nonlabens marinus rhodopsin-3 studied by static and time-resolved spectroscopy　　　　　　　　　　　　　　　
塚本 卓
日本生物物理学会第55回年会, 19 Sep. 2017, English, Poster presentation
[Domestic Conference]

好熱性バクテリアのレチナールタンパク質：発見と物性解析のこれまでとこれから　　　　　　　　　　　　　　　
塚本 卓
第54回日本生化学会北海道支部例会, 07 Jul. 2017, Japanese, Invited oral presentation
[Invited], [Domestic Conference]

Structural basis for high thermal stability and efficient optogenetic function of thermophilic rhodopsin　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第54回年会, Nov. 2016, English, Poster presentation
[Domestic Conference]

Cl--pumping photoreaction of a bacterial halide-ion pumping rhodopsin with an archaeal-type TSA motif　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第54回年会, Nov. 2016, English, Poster presentation
[Domestic Conference]

微生物型ロドプシンの安定化機構の解明と光操作への展開　　　　　　　　　　　　　　　
塚本 卓
CREST「ファイバーレス光遺伝学による高次脳機能を支える本脳機能の解明」第１回ワークショップ, Nov. 2016, Japanese, Others
[Domestic Conference]

微生物型ロドプシンの光応答性と応用技術への展開　　　　　　　　　　　　　　　
塚本 卓
新学術領域研究「人工光合成」若手育成シンポジウム〜生物から学び、応用する光反応〜, Nov. 2016, Japanese, Public symposium
[Invited], [Domestic Conference]

高度好熱菌Thermus thermophilus 由来サーモフィリックロドプシンの物性研究　　　　　　　　　　　　　　　
塚本 卓
日本生物物理学会中四国支部講演会, May 2016, Japanese, Oral presentation
[Domestic Conference]

時間分解吸収分光法で膜タンパク質の反応中間体をとらえる　　　　　　　　　　　　　　　
塚本 卓
第２回創薬標的膜タンパク質の移ろいを“み（見・診・覧）る”研究会, Oct. 2015, Japanese, Others
[Domestic Conference]

X-ray Crystal Structure of TR: Implications for High Thermal Stability and High-Performance Optogenetic Availability　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第53回年会, Sep. 2015, English, Poster presentation
[Domestic Conference]

カロテノイドを光捕集系とするレチナールタンパク質の創成と展開　　　　　　　　　　　　　　　
塚本 卓
新学術研究「人工光合成」第4回合同班会議, Jun. 2015, Japanese, Public symposium
[Domestic Conference]

Structural and Functional Characterization of Thermophilic Rhodopsin　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
新学術研究「柔らかな分子系」第8回ワークショップ「やわらか光受容分子の理解と利用に迫るブレインストーミング研究会」, Jan. 2015, Japanese, Public symposium
[Domestic Conference]

高度高熱菌由来光受容膜タンパク質ロドプシンの機能・構造研究　　　　　　　　　　　　　　　
塚本 卓
第１回創薬標的膜タンパク質の移ろいを“み（見・診・覧）る”研究会, Nov. 2014, Japanese, Others
[Domestic Conference]

Physicochemical Characterization of a Light-Driven Proton Pump from an Extreme Thermophile　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
16th International Conference on Retinal Proteins, Oct. 2014, English, Poster presentation
[International presentation]

Temperature-Dependent Irreversible Structural Transition of Thermophilic Rhodopsin　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第52回年会, Sep. 2014, English, Poster presentation
[Domestic Conference]

骨芽細胞膜タンパク質IFITM5の分子機構：翻訳後修飾，相互作用，および細胞機能　　　　　　　　　　　　　　　
塚本 卓
分子・物質合成プラットフォーム平成25年度シンポジウム, Mar. 2014, Japanese, Public symposium
[Domestic Conference]

2P233 Temperature-Dependent Irreversible Structural Transition of Thermophilic Rhodopsin(18A. Photobiology:Vision & Photoreception,Poster)
Tsukamoto Takashi, Demura Makoto, Sudo Yuki
Seibutsu Butsuri, 2014, The Biophysical Society of Japan General Incorporated Association, English
2014 - 2014

3P101 Functional significance of homotrimer formation in the Nanodisc-embedded Halorhodopsin(03. Membrane proteins,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))
Suzuki Kenshiro, Yamamoto Ayumi, Tsukamoto Takashi, Kobashigawa Yoshihiro, Uchida Takeshi, Inagaki Fuyuhiko, Demura Makoto, Ishimori Koichiro
Seibutsu Butsuri, 2014, The Biophysical Society of Japan General Incorporated Association, English
2014 - 2014

生理的温度条件下における高度好熱菌由来ロドプシン・TRの物性に関する研究　　　　　　　　　　　　　　　
塚本 卓
分子研研究会「ロドプシン研究の故きを温ねて新しきを知る」, Nov. 2013, Japanese, Public symposium
[Domestic Conference]

Thermophilic rhodopsin: The first light-driven proton pump from an extreme thermophile　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第51回年会, Oct. 2013, English, Poster presentation
[Domestic Conference]

高度好熱菌Thermus thermophilus由来微生物型ロドプシンの分光学的解析　　　　　　　　　　　　　　　
塚本 卓
2012年度生物物理学会中部支部講演会, Feb. 2013, Japanese, Oral presentation
[Domestic Conference]

3P105 Effects of homotrimer formation on chloride pump activity in membrane mimetics, Nanodisc, embedded Halorhodopsin(03. Membrane proteins,Poster)
Suzuki Kenshiro, Yamamoto Ayumi, Tsukamoto Takashi, Kobashigawa Toshihiro, Uchida Takeshi, Inagaki Fuyuhiko, Demura Makoto, Ishimori Koichiro
Seibutsu Butsuri, 2013, The Biophysical Society of Japan General Incorporated Association, English
2013 - 2013

1P243 Expression of channelrhodopsin-1 in Esherichia coli(18A. Photobiology: Vision & Photoreception,Poster)
Mori Arisa, Tsukamoto Takashi, Yagasaki Zin, Homma Michio, Ihara Kunio, Sudo Yuki
Seibutsu Butsuri, 2013, The Biophysical Society of Japan General Incorporated Association, English
2013 - 2013

1P247 Light-induced inward proton transport in a blue-shifted archaerhodopsin-3 mutant(18A. Photobiology: Vision & Photoreception,Poster)
Inoue Keiichi, Tsukamoto Takashi, Yagasaki Jin, Shimono Kazumi, Miyauchi Seiji, Hayashi Shigehiko, Kandori Hideki, Sudo Yuki
Seibutsu Butsuri, 2013, The Biophysical Society of Japan General Incorporated Association, English
2013 - 2013

1P244 Thermophilic rhodopsin: The first light-driven proton pump from an extreme thermophile(18A. Photobiology: Vision & Photoreception,Poster)
Tsukamoto Takashi, Sudo Yuki
Seibutsu Butsuri, 2013, The Biophysical Society of Japan General Incorporated Association, English
2013 - 2013

2P244 Analysis of Cl^- release and uptake steps of light-driven Cl^- pump Natronomonas pharaonis halorhodopsin(18A. Photobiology: Vision & Photoreception,Poster)
Kikukawa Takashi, Kusakabe Chikara, Kokubo Asami, Tsukamoto Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Ihara Kunio, Kamo Naoki, Demura Makoto
Seibutsu Butsuri, 2013, The Biophysical Society of Japan General Incorporated Association, English
2013 - 2013

Homo-trimeric structure of Gloeobacter rhodopsin is regulated by the protonation state of Asp121, a counterion of the protonated Schiff base　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
15th International Conference on Retinal Proteins, Oct. 2012, English, Poster presentation
[International presentation]

Homotrimeric assembly of a cyanobacterial ion-pump Gloeobacter rhodopsin　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第50回年会, Sep. 2012, English, Poster presentation
[Domestic Conference]

DDM可溶化系におけるGloeobacter rhodopsinの多量体形成に関する研究　　　　　　　　　　　　　　　
塚本 卓
日本生物物理学会2011年度北海道支部例会, Mar. 2012, Japanese, Oral presentation
[Domestic Conference]

Study of the oligomeric assembly of Gloeobacter rhodopsin in DDM solution　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
9th Japan-Korea Bilateral Symposium on Biological NMR, Mar. 2012, English, Others
[International presentation]

2PT179 Homo-trimeric assembly of a cyanobacterial ion-pump Gloeobacter rhodopsin(The 50th Annual Meeting of the Biophysical Society of Japan)
Tsukamoto Takashi, Kikukawa Takashi, Kamiya Masakatsu, Aizawa Tomoyasu, Kawano Keiichi, Jung Kwang-Hwan, Kamo Naoki, Demura Makoto
Seibutsu Butsuri, 2012, The Biophysical Society of Japan General Incorporated Association, English
2012 - 2012

The role of cysteines in the first transmembrane domain of IFITM5 for the intermolecular interaction with FKBP11　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
第34回日本分子生物学会年会, Dec. 2011, Japanese, Oral presentation
[Domestic Conference]

Role of Ser171 for the stabilization of NpHR trimer　　　　　　　　　　　　　　　
塚本 卓
日本生物物理学会第49回年会, Sep. 2011, English, Oral presentation
[Domestic Conference]

Trimerization of halorhodopsin by the aromatic-interemolecular interaction and its functional modulation in detergent system　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第48回年会, Sep. 2010, English, Poster presentation
[Domestic Conference]

Trimer formation of N. pharaonis halorhodopsin is stabilized by the intermolecular interaction among Phe150 residues and leads to functional modulation in detergent solution　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
14th International Conference on Retinal Proteins, Aug. 2010, English, Poster presentation
[International presentation]

Expression, purification and characterization of osteoblast-specific membrane protein IFITM5 in detergent solubilized system　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
第10回日本蛋白質科学会年会, Jun. 2010, Japanese, Poster presentation
[Domestic Conference]

膜タンパク質IFITM5の立体構造研究；溶液NMR法による立体構造解析に向けた試料調製　　　　　　　　　　　　　　　
塚本 卓
第48回NMR討論会, Nov. 2009, Japanese, Poster presentation
[Domestic Conference]

ハロロドプシンNpHRの三量体構造はF150残基の対称性によって安定化される　　　　　　　　　　　　　　　
塚本 卓
日本生物物理学会第47回年会, Oct. 2009, English, Poster presentation
[Domestic Conference]

Structural study of IFITM5, a human double transmembrane protein; sample preparation for NMR analysis　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第47回年会, Oct. 2009, English, Poster presentation
[Domestic Conference]

Structural study of halorhodopsin: What makes its trimeric assembly stable in the DDM-solubilized system?　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
7th Japan-Korea Bilateral Symposium on Biological NMR, Jul. 2009, English, Others
[International presentation]

Molecular dynamics of photo-excited halorhodopsin in the monomer and trimer states　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
Hokkaido University – Mahidol University Joint Symposium, May 2009, English, Others
[International presentation]

Molecular interaction and functional modulation of light-driven anion pump - halorhodopsin　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
第9回日本蛋白質科学会年会, May 2009, Japanese, Poster presentation
[Domestic Conference]

Molecular dynamics of photo-excited halorhodopsin in the monomer and trimer states　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
揺らぎと生体機能第2回公開シンポジウム, Mar. 2009, Japanese, Public symposium
[Domestic Conference]

Is the photoactivity of halorhodopsin modulated by the assembly of homotrimer?” 3P-097　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
日本生物物理学会第46回年会, Dec. 2008, English, Poster presentation
[Domestic Conference]

Trimer assembly of Natronomonas pharaonis halorhodopsin, NpHR, in lipid bilayer and detergent　　　　　　　　　　　　　　　
TSUKAMOTO Takashi
13th International Conference on Retinal Proteins, Jun. 2008, English, Poster presentation
[International presentation]

ハロロドプシン三量体の界面活性剤中での解離　　　　　　　　　　　　　　　
塚本 卓
日本生物物理学会第45回年会, Dec. 2007, Japanese, Oral presentation
[Domestic Conference]

Chemistry　　　　　　　　　　　　　　　
Hokkaido University
2022 - Present

SDGs and Life Science　　　　　　　　　　　　　　　
Hokkaido University
2020 - Present

生体高分子学実験　　　　　　　　　　　　　　　
北海道大学
2017 - Present

実験生物科学　　　　　　　　　　　　　　　
北海道大学
2018 - 2023

物理学　　　　　　　　　　　　　　　
北海道大学
2018 - 2021

細胞構造科学　　　　　　　　　　　　　　　
北海道大学
2017 - 2018

コミュニケーション入門　　　　　　　　　　　　　　　
岡山大学

薬学基本実習　　　　　　　　　　　　　　　
岡山大学

薬学基礎実習　　　　　　　　　　　　　　　
岡山大学

物理学　　　　　　　　　　　　　　　
岡山大学

2007 - Present
THE BIOPHYSICAL SOCIETY OF JAPAN　　　　　　　　　　　　　　　

アニオンチャネルロドプシンの動的なイオン選択性メカニズムの解明
科学研究費助成事業 基盤研究(C)
01 Apr. 2023 - 31 Mar. 2026
塚本 卓
日本学術振興会, 基盤研究(C), 北海道大学, 23K05675

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
01 Apr. 2022 - 31 Mar. 2025
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)
01 Apr. 2022 - 31 Mar. 2025
菊川 峰志, 宮内 正二, 塚本 卓, 海野 雅司
日本学術振興会, 基盤研究(B), 北海道大学, 22H02579

全長型アニオンチャネルロドプシンのイオン輸送機能：C末端ドメインの役割について
科学研究費助成事業 基盤研究(C)
01 Apr. 2022 - 31 Mar. 2025
出村 誠, 塚本 卓
日本学術振興会, 基盤研究(C), 北海道大学, 22K06120

天然全長型アニオンチャネルロドプシンの見過ごされてきた分子機能
2022年度秋山記念生命科学研究助成金
Sep. 2022 - Mar. 2023
公益財団法人秋山記念生命科学振興財団, 北海道大学大学院先端生命科学研究院, Principal investigator

トライアル研究支援制度　　　　　　　　　　　　　　　
トライアル研究支援制度
Oct. 2021 - Mar. 2022
北海道大学, 北海道大学, Principal investigator

生体分子中の窒素の特性を、中性子回折と計算化学により解明する　　　　　　　　　　　　　　　
科学研究費補助金
Feb. 2019 - Mar. 2021
尾瀬 農之
日本学術振興会, Competitive research funding

イオン輸送型ロドプシンのアミノ酸変異による輸送方式の変換とその分子機構の解明　　　　　　　　　　　　　　　
KAKENHI
Apr. 2018 - Mar. 2020
TSUKAMOTO Takashi
JSPS, Principal investigator, Competitive research funding

Investigation, modification and utilization of the retinal proteins
Grants-in-Aid for Scientific Research
01 Apr. 2015 - 31 Mar. 2018
Sudo Yuki, TSUKAMOTO Takashi
Retinal protein, also called "rhodopsin", has a vitamin-A aldehyde as a chromophore. It is widely distributed in the three biological domains (animals, bacteria, archaea), and is responsible for various light-dependent functions. In addition to such biological interests, recently, the new technology called "optogenetics" which is a method for controlling biological activities by light, has been established as a collaborative work with the retinal proteins. In this research, based on the background, we investigated the retinal proteins fundamentally by using various methods. Then we modified the molecular properties to develop the novel optogenetics tools that can be widely utilized for scientific research.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Okayama University, 15H04363

光駆動細胞内向きプロトン輸送タンパク質の分子機構の解明と応用技術　　　　　　　　　　　　　　　
若手研究人材・ネットワーク育成補助金（ノースタレント補助金）
Jun. 2017 - Mar. 2018
塚本 卓
ノーステック財団, Principal investigator, Competitive research funding

スタートアップ経費
Apr. 2017 - Mar. 2018
塚本 卓
北海道大学, Principal investigator, Competitive research funding

高度好熱菌ロドプシンにおける構造安定性の追求（研究課題番号：15K18519）　　　　　　　　　　　　　　　
Grants-in-Aid for Scientific Research (KAKEN)
Apr. 2015 - Mar. 2018
TSUKAMOTO Takashi
JSPS, Principal investigator, Competitive research funding

Time-resolved infrared spectroscopy of the thermophilic rhodopsin under various pressures and temperatures
Grants-in-Aid for Scientific Research
28 Aug. 2015 - 31 Mar. 2017
Kuroi Kunisato, Tsukamoto Takashi
We investigated the photoreaction of the thermophilic rhodopsin (TR) by time-resolved Fourier transform infrared spectroscopy (TR-FTIR) under various temperature and pressure conditions to thermodynamically characterize its photoreaction. First, from the TR-FTIR measurement of TR and its mutants at 50 degree, we revealed the intramolecular proton transfer mechanism of TR. Second, from the TR-FTIR measurement of TR at various temperatures from 30 to 70 degrees, we found that the photoreaction mechanism of TR seemed to switch at about 40 degree. Finally, we developed the measurement system of high-pressure TR-FTIR, and applied it to the photoreaction of the bacteriorhodopsin (BR). We succeeded in measuring the TR-FTIR spectra of BR under high-pressure for the first time. However, due to the bad signal to noise ratio of the system, unfortunately we couldn’t apply that system to TR by the end of this project.
Japan Society for the Promotion of Science, Grant-in-Aid for Research Activity Start-up, 15H06837

カロテノイド結合型ロドプシンの２光子吸収による活性化の可能性調査　　　　　　　　　　　　　　　
Research for Specific Issues, PRESTO
Oct. 2016 - Mar. 2017
TSUKAMOTO Takashi
JST, Principal investigator, Competitive research funding

時間分解赤外分光法によるレチナールタンパク質の構造ダイナミクスの解析　　　　　　　　　　　　　　　
平成28年度自然科学研究機構分子科学研究所共同利用研究（前期）
Apr. 2016 - Sep. 2016
塚本 卓
自然科学研究機構分子科学研究所, Principal investigator, Competitive research funding

若手研究者スタートアップ研究支援事業
Jul. 2014 - Jun. 2016
塚本 卓
岡山大学, Principal investigator, Competitive research funding

時間分解赤外分光法によるレチナールタンパク質の構造ダイナミクスの解析　　　　　　　　　　　　　　　
平成27年度自然科学研究機構分子科学研究所共同利用研究（後期）
Oct. 2015 - Mar. 2016
塚本 卓
自然科学研究機構分子科学研究所, Principal investigator, Competitive research funding