Publications by authors named "Sahoko Tomida"

9 Publications

  • Page 1 of 1

Inverse Hydrogen-Bonding Change Between the Protonated Retinal Schiff Base and Water Molecules upon Photoisomerization in Heliorhodopsin 48C12.

J Phys Chem B 2021 08 22;125(30):8331-8341. Epub 2021 Jul 22.

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.

Heliorhodopsin (HeR) is a new class of the rhodopsin family discovered in 2018 through functional metagenomic analysis (named 48C12). Similar to typical microbial rhodopsins, HeR possesses seven transmembrane (TM) α-helices and an all--retinal covalently bonded to the lysine residue on TM7 via a protonated Schiff base. Remarkably, the HeR membrane topology is inverted compared with that of typical microbial rhodopsins. The X-ray crystal structure of HeR 48C12 was elucidated after the first report on a HeR variant from SG8-52-1, which revealed the water-mediated hydrogen-bonding network connected to the Schiff base region in the cytoplasmic side. Herein, low-temperature light-induced FTIR spectroscopic analyses of HeR 48C12 and N isotopically labeled proteins were used to elucidate the structural changes during retinal photoisomerization. N-D stretching vibrations of the protonated retinal Schiff base (PRSB) at 2286 and 2302 cm in the dark state, and 2239 and 2252 cm in the K intermediate were observed. The frequency changes indicated that the hydrogen bond of PRSB strengthens upon photoisomerization in HeR. Moreover, O-D stretching vibration frequencies of the internal water molecules indicate that the hydrogen-bonding strength decreases concomitantly. Therefore, the PRSB hydrogen bond responds to photoisomerization in an opposite way to the hydrogen-bonding network involving water molecules. No frequency changes of the indole N-H or N-D stretching vibrations of tryptophan residues were observed upon photoisomerization, suggesting that all tryptophan residues in the HeR 48C12 maintained the hydrogen-bonding strengths in the K intermediate. These results provide insights into the molecular mechanism of the energy storage and propagation upon retinal photoisomerization in HeR.
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http://dx.doi.org/10.1021/acs.jpcb.1c01907DOI Listing
August 2021

Mechanism of Inward Proton Transport in an Antarctic Microbial Rhodopsin.

J Phys Chem B 2020 06 5;124(24):4851-4872. Epub 2020 Jun 5.

Department of Physics, University of Guelph, 50 Stone Rd. E., Guelph, Ontario N1G 2W1, Canada.

Although the outward-directed proton transport across biological membranes is well studied and its importance for bioenergetics is clearly understood, inward-directed light-driven proton pumping by microbial rhodopsins has remained a mystery both physiologically and mechanistically. A new family of Antarctic rhodopsins, which is a subgroup within a novel class of schizorhodopsins reported recently, includes a member, denoted as AntR, which proved amenable to extensive characterization with experiments and computation. Phylogenetic analyses identify AntR as distinct from the well-studied microbial rhodopsins that function as outward-directed ion pumps, and bioinformatics sequence analyses reveal amino acid substitutions at conserved sites essential for outward proton pumping. Modeling and numerical simulations of AntR, combined with advanced analyses using the graph theory and centrality measures from social sciences, identify the dynamic three-dimensional network of hydrogen-bonded water molecules and amino acid residues that function as communication hubs in AntR. This network undergoes major rearrangement upon retinal isomerization, showing important changes in the connectivity of the active center, retinal Schiff base, to the opposing sides of the membrane, as required for proton transport. Numerical simulations and experimental studies of the photochemical cycle of AntR by spectroscopy and site-directed mutagenesis allowed us to identify pathways that could conduct protons in the direction opposite to that commonly known for outward-directed pumps.
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http://dx.doi.org/10.1021/acs.jpcb.0c02767DOI Listing
June 2020

Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H pumps.

Sci Adv 2020 04 10;6(15):eaaz2441. Epub 2020 Apr 10.

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.

Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H pump. The mutation of a cytoplasmic glutamate inhibited inward H transport, suggesting that it functions as a cytoplasmic H acceptor. The function, trimeric structure, and H transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H pumping microbial rhodopsins, implying that they evolved convergently. The inward H pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.
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http://dx.doi.org/10.1126/sciadv.aaz2441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148096PMC
April 2020

Infrared spectroscopic analysis on structural changes around the protonated Schiff base upon retinal isomerization in light-driven sodium pump KR2.

Biochim Biophys Acta Bioenerg 2020 07 17;1861(7):148190. Epub 2020 Mar 17.

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan; OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan. Electronic address:

Krokinobacter rhodopsin 2 (KR2) was discovered as the first light-driven sodium pumping rhodopsin (NaR) in 2013, which contains unique amino acid residues on C-helix (N112, D116, and Q123), referred to as an NDQ motif. Based on the recent X-ray crystal structures of KR2, the sodium transport pathway has been investigated by various methods. However, due to complicated structural information around the protonated Schiff base (PRSB) region in the dark state and lack of structural information in the intermediates with sodium bound in KR2, detailed sodium pump mechanism is still unclear. Here we applied comprehensive low-temperature light-induced difference FTIR spectroscopy on isotopically labeled KR2 WT and site-directed mutant proteins (N112A, D116E, R109A, and R109K). We assigned the N-D stretching vibration of the PRSB at 2095 cm and elucidate the hydrogen bonding interaction with D116 (a counter ion for the PRSB). We also assigned strongly hydrogen-bonded water (2333 cm) near R109 and D251, and found that presence of a positive charge at the position of R109 is prerequisite for the pumping function of KR2.
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http://dx.doi.org/10.1016/j.bbabio.2020.148190DOI Listing
July 2020

Crystal structure of heliorhodopsin.

Nature 2019 10 25;574(7776):132-136. Epub 2019 Sep 25.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics. They are widely present in bacteria, archaea, algae and algal viruses. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the β-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.
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http://dx.doi.org/10.1038/s41586-019-1604-6DOI Listing
October 2019

Red-shifting mutation of light-driven sodium-pump rhodopsin.

Nat Commun 2019 04 30;10(1):1993. Epub 2019 Apr 30.

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya, 466-8555, Japan.

Microbial rhodopsins are photoreceptive membrane proteins that transport various ions using light energy. While they are widely used in optogenetics to optically control neuronal activity, rhodopsins that function with longer-wavelength light are highly demanded because of their low phototoxicity and high tissue penetration. Here, we achieve a 40-nm red-shift in the absorption wavelength of a sodium-pump rhodopsin (KR2) by altering dipole moment of residues around the retinal chromophore (KR2 P219T/S254A) without impairing its ion-transport activity. Structural differences in the chromophore of the red-shifted protein from that of the wildtype are observed by Fourier transform infrared spectroscopy. QM/MM models generated with an automated protocol show that the changes in the electrostatic interaction between protein and chromophore induced by the amino-acid replacements, lowered the energy gap between the ground and the first electronically excited state. Based on these insights, a natural sodium pump with red-shifted absorption is identified from Jannaschia seosinensis.
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http://dx.doi.org/10.1038/s41467-019-10000-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491443PMC
April 2019

A distinct abundant group of microbial rhodopsins discovered using functional metagenomics.

Nature 2018 06 20;558(7711):595-599. Epub 2018 Jun 20.

Faculty of Biology, Technion Israel Institute of Technology, Haifa, Israel.

Many organisms capture or sense sunlight using rhodopsin pigments, which are integral membrane proteins that bind retinal chromophores. Rhodopsins comprise two distinct protein families , type-1 (microbial rhodopsins) and type-2 (animal rhodopsins). The two families share similar topologies and contain seven transmembrane helices that form a pocket in which retinal is linked covalently as a protonated Schiff base to a lysine at the seventh transmembrane helix. Type-1 and type-2 rhodopsins show little or no sequence similarity to each other, as a consequence of extensive divergence from a common ancestor or convergent evolution of similar structures . Here we report a previously unknown and diverse family of rhodopsins-which we term the heliorhodopsins-that we identified using functional metagenomics and that are distantly related to type-1 rhodopsins. Heliorhodopsins are embedded in the membrane with their N termini facing the cell cytoplasm, an orientation that is opposite to that of type-1 or type-2 rhodopsins. Heliorhodopsins show photocycles that are longer than one second, which is suggestive of light-sensory activity. Heliorhodopsin photocycles accompany retinal isomerization and proton transfer, as in type-1 and type-2 rhodopsins, but protons are never released from the protein, even transiently. Heliorhodopsins are abundant and distributed globally; we detected them in Archaea, Bacteria, Eukarya and their viruses. Our findings reveal a previously unknown family of light-sensing rhodopsins that are widespread in the microbial world.
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http://dx.doi.org/10.1038/s41586-018-0225-9DOI Listing
June 2018

Hydrogen-bonding network at the cytoplasmic region of a light-driven sodium pump rhodopsin KR2.

Biochim Biophys Acta Bioenerg 2018 09 28;1859(9):684-691. Epub 2018 May 28.

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan; OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan. Electronic address:

Light-driven sodium-pumping rhodopsins are able to actively transport sodium ions. Structure/function studies of Krokinobacter eikastus rhodopsin 2 (KR2) identified N61 and G263 at the cytoplasmic surface constituting the "Ion-selectivity filter" for sodium ions, while retinal Schiff base acts as the light "Switch and Gate" for transport of sodium ions. Q123 is located between the two regions, and plays an important role for the pump function, which was implicated by functional, spectroscopic, X-ray crystallographic and computational studies. According to the atomic structure of KR2, Q123 is involved in the hydrogen-bonding network at the cytoplasmic region, together with S64, protein-bound waters, and peptide carbonyl of K255 bound to the chromophore. To gain the detailed structural information around Q123, here we compared light-induced difference Fourier-transform infrared (FTIR) spectra at 77 K between the wild-type (WT) and mutant proteins of KR2, such as Q123A, Q123V, and S64A. The obtained spectra were very similar between WT and these mutants, whereas the observed mutation effects enabled us to identify vibrations of the hydrogen-bonding network at the Q123 and S64 region. This is unique for KR2, not for the corresponding mutations in a light-driven proton-pump bacteriorhodopsin (BR). Hydrogen-bonding alteration is absent for the mutants of KR2, suggesting that proper inter-helical connectivity of helices B, C, and G is important for protein structural changes for sodium-pump function, which is controlled by the region around Q123.
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http://dx.doi.org/10.1016/j.bbabio.2018.05.017DOI Listing
September 2018

Long-distance perturbation on Schiff base-counterion interactions by His30 and the extracellular Na-binding site in Krokinobacter rhodopsin 2.

Phys Chem Chem Phys 2018 Mar;20(13):8450-8455

Graduate School of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, Japan.

Krokinobacter rhodopsin 2 (KR2), a light-driven Na+ pump, is a dual-functional protein, pumping protons in the absence of Na+ when K+ or larger alkali metal ions are present. A specific mutation in helix A near the extracellular Na+ binding site, H30A, eliminates its proton pumping ability. We induced structural changes in H30A by altering the alkali metal ion bound at the extracellular binding site, and observed a strong electrostatic interaction between the Schiff base and counterion and torsion around the Schiff base as revealed by solid-state nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR) spectroscopies. The strong interaction when His30 was absent and no ion bound at the extracellular binding site disabled retinal reisomerization, as was shown with flash-photolysis, forming a small amount of only a K-like intermediate. This revealed why H30A lacks the proton pumping function. Long-distance perturbation of the binding site and Schiff base revealed that a non-transported ion binding at the extracellular site is essential for pumping.
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http://dx.doi.org/10.1039/c8cp00626aDOI Listing
March 2018
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