Publications by authors named "Hideki Kandori"

254 Publications

Ion transport activity and optogenetics capability of light-driven Na+-pump KR2.

PLoS One 2021 10;16(9):e0256728. Epub 2021 Sep 10.

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

KR2 from marine bacteria Krokinobacter eikastus is a light-driven Na+ pumping rhodopsin family (NaRs) member that actively transports Na+ and/or H+ depending on the ionic state. We here report electrophysiological studies on KR2 to address ion-transport properties under various electrochemical potentials of Δ[Na+], ΔpH, membrane voltage and light quality, because the contributions of these on the pumping activity were less understood so far. After transient expression of KR2 in mammalian cultured cells (ND7/23 cells), photocurrents were measured by whole-cell patch clamp under various intracellular Na+ and pH conditions. When KR2 was continuously illuminated with LED light, two distinct time constants were obtained depending on the Na+ concentration. KR2 exhibited slow ion transport (τoff of 28 ms) below 1.1 mM NaCl and rapid transport (τoff of 11 ms) above 11 mM NaCl. This indicates distinct transporting kinetics of H+ and Na+. Photocurrent amplitude (current density) depends on the intracellular Na+ concentration, as is expected for a Na+ pump. The M-intermediate in the photocycle of KR2 could be transferred into the dark state without net ion transport by blue light illumination on top of green light. The M intermediate was stabilized by higher membrane voltage. Furthermore, we assessed the optogenetic silencing effect of rat cortical neurons after expressing KR2. Light power dependency revealed that action potential was profoundly inhibited by 1.5 mW/mm2 green light illumination, confirming the ability to apply KR2 as an optogenetics silencer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0256728PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432791PMC
September 2021

Ion Transport Activity Assay for Microbial Rhodopsin Expressed in Cells.

Bio Protoc 2021 Aug 5;11(15):e4115. Epub 2021 Aug 5.

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

Microbial rhodopsins have diverse functions, including roles as light-driven ion pumps, light-gated ion channels, photosensors, and light-regulated enzymes. As the number of rhodopsin-like genes identified has increased in recent years, so has the requirement for rapid identification of their functions. The patch-clamp method is often used to investigate the ion transport mechanism of microbial rhodopsins in mammalian cells; however, this requires a dedicated system and advanced techniques. The ion transport assay using the expression system described here evaluates the ion transport capacity by monitoring the pH change in suspensions; if the target rhodopsin has a light-dependent ion transport activity, a light-dependent pH change is observed. The pH increase or decrease corresponds to proton release from the cell or proton uptake into the cell, respectively. This method can be used to evaluate ion transport capacity in a high-throughput manner using a combination of general-purpose equipment and common techniques. Graphic abstract: Schematic diagram of the ion transport assay in rhodopsin-expressing cells.
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http://dx.doi.org/10.21769/BioProtoc.4115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8376562PMC
August 2021

Microbial Rhodopsins: The Last Two Decades.

Annu Rev Microbiol 2021 Aug 3. Epub 2021 Aug 3.

Faculty of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel; email:

Microbial rhodopsins are diverse photoreceptive proteins containing a retinal chromophore and are found in all domains of cellular life and are even encoded in genomes of viruses. These rhodopsins make up two families: type 1 rhodopsins and the recently discovered heliorhodopsins. These families have seven transmembrane helices with similar structures but opposing membrane orientation. Microbial rhodopsins participate in a portfolio of light-driven energy and sensory transduction processes. In this review we present data collected over the last two decades about these rhodopsins and describe their diversity, functions, and biological and ecological roles. Expected final online publication date for the , Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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http://dx.doi.org/10.1146/annurev-micro-031721-020452DOI Listing
August 2021

Remote control of neural function by X-ray-induced scintillation.

Nat Commun 2021 07 22;12(1):4478. Epub 2021 Jul 22.

Department of Neuroscience II, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.

Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd(Al,Ga)O (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.
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http://dx.doi.org/10.1038/s41467-021-24717-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298491PMC
July 2021

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

Resonance Raman Determination of Chromophore Structures of Heliorhodopsin Photointermediates.

J Phys Chem B 2021 07 24;125(26):7155-7162. Epub 2021 Jun 24.

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.

Light is utilized as energy or information by rhodopsins (membrane proteins that contain a retinal chromophore). Heliorhodopsins (HeRs) are a new class of rhodopsins with low sequence identity (<15%) to microbial and animal rhodopsins. Their physiological roles remain unknown, although the involvement of a long-lived intermediate in the photocycle suggests a light-sensor function. Characterization of the molecular structures of the intermediates is essential to an understanding of the roles and mechanisms of HeRs. We determined the chromophore structures of the intermediates in HeR 48C12 by time-resolved resonance Raman spectroscopy and observed that the hydrogen bond of the protonated Schiff base strengthened prior to deprotonation. The chromophore is photoisomerized from the all- to the 13- form and is reisomerized in the transition from the O intermediate to the unphotolyzed state. Our results demonstrate that the chromophore structure evolves similarly to microbial rhodopsins, despite the dissimilarity in amino acid residues surrounding the chromophore.
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http://dx.doi.org/10.1021/acs.jpcb.1c04010DOI Listing
July 2021

Role of Thr82 for the unique photochemistry of TAT rhodopsin.

Biophys Physicobiol 2021 16;18:108-115. Epub 2021 Apr 16.

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

Marine bacterial TAT rhodopsin possesses the pKa of the retinal Schiff base, the chromophore, at neutral pH, and photoexcitation of the visible protonated state forms the isomerized 13- state, but reverts to the original state within 10 sec. To understand the origin of these unique molecular properties of TAT rhodopsin, we mutated Thr82 into Asp, because many microbial rhodopsins contain Asp at the corresponding position as the Schiff base counterion. A pH titration study revealed that the pKa of the Schiff base increased considerably in T82D (>10.5), and that the pKa of the counterion, which is likely to be D82, is 8.1. It was thus concluded that T82 is the origin of the neutral pKa of the Schiff base in TAT rhodopsin. The photocycle of T82D TAT rhodopsin exhibited strong pH dependence. When pH is lower than the pKa of the counterion (pH <8.1), formation of the primary K intermediate was observed by low-temperature UV-visible spectroscopy, but flash photolysis failed to monitor photointermdiates at >10 sec. The results were identical for the wild-type TAT rhodopsin. In contrast, when pH was higher than the pKa of the counterion, we observed the formation of the M intermediate, which decayed with the time constants of 3.75 ms and 12.2 sec. It is likely that the protonation state of D82 dramatically switches the photoreaction dynamics of T82D, whose duration lies between <10 sec and >10 sec. It was thus concluded that T82 is one of the determinants of the unique photochemistry of TAT rhodopsin.
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http://dx.doi.org/10.2142/biophysico.bppb-v18.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116198PMC
April 2021

Light-induced difference FTIR spectroscopy of primate blue-sensitive visual pigment at 163 K.

Biophys Physicobiol 2021 13;18:40-49. Epub 2021 Feb 13.

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

Structural studies of color visual pigments lag far behind those of rhodopsin for scotopic vision. Using difference FTIR spectroscopy at 77 K, we report the first structural data of three primate color visual pig-ments, monkey red (MR), green (MG), and blue (MB), where the batho-intermediate (Batho) exhibits photo-equilibrium with the unphotolyzed state. This photo-chromic property is highly advantageous for limited samples since the signal-to-noise ratio is improved, but may not be applicable to late intermediates, because of large structural changes to proteins. Here we report the photochromic property of MB at 163 K, where the BL intermediate, formed by the relaxation of Batho, is in photoequilibrium with the initial MB state. A comparison of the difference FTIR spectra at 77 and 163 K provided information on what happens in the process of transition from Batho to BL in MB. The coupled C=C HOOP vibration in the planer structure in MB is decoupled by distortion in Batho after retinal photoisomerization, but returns to the coupled C=C HOOP vibration in the all- chromophore in BL. The Batho formation accompanies helical structural perturbation, which is relaxed in BL. Protein-bound water molecules that form an extended water cluster near the retinal chromophore change hydrogen bonds differently for Batho and BL, being stronger in the latter than in the initial state. In addition to structural dynamics, the present FTIR spectra show no signals of protonated carboxylic acids at 77 and 163 K, suggesting that E181 is deprotonated in MB, Batho and BL.
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http://dx.doi.org/10.2142/biophysico.bppb-v18.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049776PMC
February 2021

Crystal structure of schizorhodopsin reveals mechanism of inward proton pumping.

Proc Natl Acad Sci U S A 2021 Apr;118(14)

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan;

Schizorhodopsins (SzRs), a new rhodopsin family identified in Asgard archaea, are phylogenetically located at an intermediate position between type-1 microbial rhodopsins and heliorhodopsins. SzRs work as light-driven inward H pumps as xenorhodopsins in bacteria. Although E81 plays an essential role in inward H release, the H is not metastably trapped in such a putative H acceptor, unlike the other H pumps. It remains elusive why SzR exhibits different kinetic behaviors in H release. Here, we report the crystal structure of SzR AM_5_00977 at 2.1 Å resolution. The SzR structure superimposes well on that of bacteriorhodopsin rather than heliorhodopsin, suggesting that SzRs are classified with type-1 rhodopsins. The structure-based mutagenesis study demonstrated that the residues N100 and V103 around the β-ionone ring are essential for color tuning in SzRs. The cytoplasmic parts of transmembrane helices 2, 6, and 7 are shorter than those in the other microbial rhodopsins, and thus E81 is located near the cytosol and easily exposed to the solvent by light-induced structural change. We propose a model of untrapped inward H release; H is released through the water-mediated transport network from the retinal Schiff base to the cytosol by the side of E81. Moreover, most residues on the H transport pathway are not conserved between SzRs and xenorhodopsins, suggesting that they have entirely different inward H release mechanisms.
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http://dx.doi.org/10.1073/pnas.2016328118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040798PMC
April 2021

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin.

Elife 2021 03 23;10. Epub 2021 Mar 23.

RIKEN SPring-8 Center, Hyogo, Japan.

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.
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http://dx.doi.org/10.7554/eLife.62389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987342PMC
March 2021

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design.

Commun Biol 2021 03 19;4(1):362. Epub 2021 Mar 19.

RIKEN Center for Advanced Intelligence Project, Tokyo, Japan.

Microbial rhodopsins are photoreceptive membrane proteins, which are used as molecular tools in optogenetics. Here, a machine learning (ML)-based experimental design method is introduced for screening rhodopsins that are likely to be red-shifted from representative rhodopsins in the same subfamily. Among 3,022 ion-pumping rhodopsins that were suggested by a protein BLAST search in several protein databases, the ML-based method selected 65 candidate rhodopsins. The wavelengths of 39 of them were able to be experimentally determined by expressing proteins with the Escherichia coli system, and 32 (82%, p = 7.025 × 10) actually showed red-shift gains. In addition, four showed red-shift gains >20 nm, and two were found to have desirable ion-transporting properties, indicating that they would be potentially useful in optogenetics. These findings suggest that data-driven ML-based approaches play effective roles in the experimental design of rhodopsin and other photobiological studies. (141/150 words).
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http://dx.doi.org/10.1038/s42003-021-01878-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979833PMC
March 2021

TAT Rhodopsin Is an Ultraviolet-Dependent Environmental pH Sensor.

Biochemistry 2021 03 15;60(12):899-907. Epub 2021 Mar 15.

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

In many rhodopsins, the retinal Schiff base p remains very high, ensuring Schiff base protonation captures visible light. Nevertheless, recently we found that TAT rhodopsin contains protonated and unprotonated forms at physiological pH. The protonated form displays a unique photochemical behavior in which the primary K intermediate returns to the original state within 10 s, and the lack of photocycle completion poses questions about the functional role of TAT rhodopsin. Here we studied the molecular properties of the protonated and unprotonated forms of the Schiff base in TAT rhodopsin. We confirmed no photointermediate formation at >10 s for the protonated form of TAT rhodopsin in microenvironments such as detergents, nanodiscs, and liposomes. In contrast, the unprotonated form features a very long photocycle with a time constant of 15 s. A low-temperature study revealed that the primary reaction of the unprotonated form is all- to 13- photoisomerization, which is usual, but with a proton transfer reaction occurring at 77 K, which is unusual. The active intermediate contains the unprotonated Schiff base as well as the resting state. Electrophysiological measurements excluded ion-transport activity for TAT rhodopsin, while transient outward proton movement only at an alkaline extracellular pH indicates that TAT rhodopsin senses the extracellular pH. On the basis of the findings presented here, we propose that TAT rhodopsin is an ultraviolet (UV)-dependent environmental pH sensor in marine bacteria. At acidic pH, absorbed visible light energy is quickly dissipated into heat without any function. In contrast, when the environmental pH becomes high, absorption of UV/blue light yields formation of the long-lived intermediates, possibly driving the signal transduction cascade in marine bacteria.
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http://dx.doi.org/10.1021/acs.biochem.0c00951DOI Listing
March 2021

Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens.

Commun Biol 2021 02 23;4(1):235. Epub 2021 Feb 23.

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Nagoya, Japan.

Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.
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http://dx.doi.org/10.1038/s42003-021-01755-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902849PMC
February 2021

Molecular Properties and Optogenetic Applications of Enzymerhodopsins.

Adv Exp Med Biol 2021 ;1293:153-165

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Nagoya, Japan.

The cyclic nucleotides cAMP and cGMP are ubiquitous secondary messengers that regulate multiple biological functions including gene expression, differentiation, proliferation, and cell survival. In sensory neurons, cyclic nucleotides are responsible for signal modulation, amplification, and encoding. For spatial and temporal manipulation of cyclic nucleotide dynamics, optogenetics have a great advantage over pharmacological approaches. Enzymerhodopsins are a unique family of microbial rhodopsins. These molecules are made up of a membrane-embedded rhodopsin domain, which binds an all trans-retinal to form a chromophore, and a cytoplasmic water-soluble catalytic domain. To date, three kinds of molecules have been identified from lower eukaryotes such as fungi, algae, and flagellates. Among these, histidine kinase rhodopsin (HKR) is a light-inhibited guanylyl cyclase. Rhodopsin GC (Rh-GC) functions as a light-activated guanylyl cyclase, while rhodopsin PDE (Rh-PDE) functions as a light-activated phosphodiesterase that degrades cAMP and cGMP. These enzymerhodopsins have great potential in optogenetic applications for manipulating the intracellular cyclic nucleotide dynamics of living cells. Here we introduce the molecular function and applicability of these molecules.
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http://dx.doi.org/10.1007/978-981-15-8763-4_9DOI Listing
February 2021

History and Perspectives of Ion-Transporting Rhodopsins.

Authors:
Hideki Kandori

Adv Exp Med Biol 2021 ;1293:3-19

Department of Life Science and Applied Chemistry & OptoBioTechnology Research Center, Nagoya Institute of Technology, Nagoya, Japan.

The first light-sensing proteins used in optogenetics were rhodopsins. The word "rhodopsin" originates from the Greek words "rhodo" and "opsis," indicating rose and sight, respectively. Although the classical meaning of rhodopsin is the red-colored pigment in our eyes, the modern meaning of rhodopsin encompasses photoactive proteins containing a retinal chromophore in animals and microbes. Animal and microbial rhodopsins possess 11-cis and all-trans retinal, respectively, to capture light in seven transmembrane α-helices, and photoisomerizations into all-trans and 13-cis forms, respectively, initiate each function. We are able to find ion-transporting proteins in microbial rhodopsins, such as light-gated channels and light-driven pumps, which are the main tools in optogenetics. In this chapter, historical aspects and molecular properties of rhodopsins are introduced. In the first part, "what is rhodopsin?", general introduction of rhodopsin is presented. Then, molecular mechanism of bacteriorodopsin, a light-driven proton pump and the best-studied microbial rhodopsin, is described. In the section of channelrhodopsin, the light-gated ion channel, molecular properties, and several variants are introduced. As the history has proven, understanding the molecular mechanism of microbial rhodopsins is a prerequisite for useful functional design of optogenetics tools in future.
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http://dx.doi.org/10.1007/978-981-15-8763-4_1DOI Listing
February 2021

Expression analysis of microbial rhodopsin-like genes in Guillardia theta.

PLoS One 2020 3;15(12):e0243387. Epub 2020 Dec 3.

Life Science and Applied Chemistry, Graduate School of Engineering, Nagoya Institute of Technology, Nagoya, Japan.

The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243387PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7714340PMC
January 2021

Gate-keeper of ion transport-a highly conserved helix-3 tryptophan in a channelrhodopsin chimera, C1C2/ChRWR.

Biophys Physicobiol 2020 9;17:59-70. Epub 2020 Jun 9.

The Institute for Solid State Physics, The University of Tokyo, Kashiwa, Chiba 277-8581, Japan.

Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all- retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown. Here, we replaced the W163 (BR W86 counterpart) of a channelrhodopsin (ChR), C1C2/ChRWR, which is a chimera between ChR1 and 2, with a smaller aromatic residue, Phe to verify its role in the ion transport. Under whole-cell patch clamp recordings from the ND7/23 cells that were transfected with the DNA plasmid coding human codon optimized C1C2/ChRWR (WR) or its W163F mutant (WR-W163F), the photocurrents were evoked by a pulsatile light at 475 nm. The ion-transporting activity of WR was strongly altered by the W163F mutation in 3 points: (1) the H leak at positive membrane potential ( ) and its light-adaptation, (2) the attenuation of cation channel activity and (3) the manifestation of outward H pump activity. All of these results strongly suggest that W163 has a role in stabilizing the structure involved in the gating-on and -off of the cation channel, the role of "gate keeper". We can attribute the attenuation of cation channel activity to the incomplete gating-on and the H leak to the incomplete gating-off.
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http://dx.doi.org/10.2142/biophysico.BSJ-2020007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593130PMC
June 2020

Structural insights into the mechanism of rhodopsin phosphodiesterase.

Nat Commun 2020 11 5;11(1):5605. Epub 2020 Nov 5.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, 113-0033, Japan.

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.
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http://dx.doi.org/10.1038/s41467-020-19376-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644710PMC
November 2020

Structural basis for unique color tuning mechanism in heliorhodopsin.

Biochem Biophys Res Commun 2020 12 18;533(3):262-267. Epub 2020 Sep 18.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, 113-0033, Japan. Electronic address:

Microbial rhodopsins comprise an opsin protein with seven transmembrane helices and a retinal as the chromophore. An all-trans retinal is covalently bonded to a lysine residue through the retinal Schiff base (RSB) and stabilized by a negatively charged counterion. The distance between the RSB and counterion is closely related to the light energy absorption. However, in heliorhodopsin-48C12 (HeR-48C12), while E107 acts as the counterion, E107D mutation exhibits an identical absorption spectrum to the wild-type, suggesting that the distance does not affect its absorption spectra. Here we present the 2.6 Å resolution crystal structure of the Thermoplasmatales archaeon HeR E108D mutant, which also has an identical absorption spectrum to the wild-type. The structure revealed that D108 does not form a hydrogen bond with the RSB, and its counterion interaction becomes weaker. Alternatively, the serine cluster, S78, S112, and S238 form a distinct interaction network around the RSB. The absorption spectra of the E to D and S to A double mutants suggested that S112 influences the spectral shift by compensating for the weaker counterion interaction. Our structural and spectral studies have revealed the unique spectral shift mechanism of HeR and clarified the physicochemical properties of HeRs.
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http://dx.doi.org/10.1016/j.bbrc.2020.06.124DOI Listing
December 2020

Zinc Binding to Heliorhodopsin.

J Phys Chem Lett 2020 Oct 28;11(20):8604-8609. Epub 2020 Sep 28.

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

Heliorhodopsin (HeR), a recently discovered new rhodopsin family, has an inverted membrane topology compared to animal and microbial rhodopsins, and no ion-transport activity. The slow photocycle of HeRs suggests a light-sensor function, although the function remains unknown. HeRs exhibit no specific binding of monovalent cations or anions. Despite this, ATR-FTIR spectroscopy in the present study demonstrates binding of Zn to HeR from (TaHeR). The biding of Zn to 0.2 mM is accompanied by helical structural perturbations without altering its color. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn-binding. Similar results were obtained for HeR 48C12. These findings suggest a possible modification of HeR function by Zn.
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http://dx.doi.org/10.1021/acs.jpclett.0c02383DOI Listing
October 2020

ATP binding promotes light-induced structural changes to the protein moiety of cryptochrome 1.

Photochem Photobiol Sci 2020 Oct;19(10):1326-1331

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

Cryptochromes (CRYs) are blue-light receptors involved in photomorphogenesis in plants. Flavin adenine dinucleotide (FAD) is one of the chromophores of cryptochromes; its resting state oxidized form is converted into a signalling state neutral semiquionod radical (FADH˙) form. Studies have shown that cryptochrome 1 from Arabidopsis thaliana (AtCRY1) can bind ATP at its photolyase homology region (PHR), resulting in accumulation of FADH˙ form. This study used light-induced difference Fourier transform infrared spectroscopy to investigate how ATP influences structural changes in AtCRY1-PHR during the photoreaction. In the presence of ATP, there were large changes in the signals from the protein backbone compared with in the absence of ATP. The deprotonation of a carboxylic acid was observed only in the presence of ATP; this was assigned as aspartic acid (Asp) 396 through measurement of Asp to glutamic acid mutants. This corresponds to the protonation state of Asp396 estimated from the reported pKa values of Asp396; that is, the side chain of Asp396 is deprotonated and protonated for the ATP-free and -bound forms, respectively, in our experimental condition at pH8. Therefore, Asp396 acts a proton donor to FAD when it is ptotonated. It was indicated that the protonation/deprotination process of Asp396 is correlated with the accunumulation of FADH˙ and protein conformational changes.
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http://dx.doi.org/10.1039/d0pp00003eDOI Listing
October 2020

Disruption of Hydrogen-Bond Network in Rhodopsin Mutations Cause Night Blindness.

J Mol Biol 2020 09 11;432(19):5378-5389. Epub 2020 Aug 11.

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:

Rhodopsin is the photosensitive protein, which binds to 11-cis-retinal as its chromophore. In the dark, rhodopsin exists as a stable complex between the opsin moiety and 11-cis-retinal. The absorption of a light photon converts 11-cis-retinal to all-trans-retinal and initiates our vision. As a result, the increase in the rate of dark activation of rhodopsin reduces its photosensitivity resulting in night blindness. The mutations, G90D and T94I are night blindness-causing mutations that exhibit completely different physicochemical characteristics associated with the dark activation of rhodopsin, such as a high rate of thermal isomerization of 11-cis-retinal and a slow pigment regeneration. To elucidate the molecular mechanism by which G90D and T94I mutations affect rhodopsin dark activation and regeneration, we performed light-induced difference FTIR spectroscopy on dark and primary photo-intermediate states of G90D and T94I mutants. The FTIR spectra clearly show that both charged G90D and hydrophobic T94I mutants alter the H-bond network at the Schiff base region of the chromophore, which weakens the electrostatic interaction with Glu113 counterion. Our results further show an altered water-mediated H-bond network around the central transmembrane region of mutant rhodopsin, which is reminiscent of the active Meta-II state. This altered water-mediated H-bond network may cause thermal isomerization of the chromophore and facilitate rhodopsin dark activation.
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http://dx.doi.org/10.1016/j.jmb.2020.08.006DOI Listing
September 2020

Active Learning of Bayesian Linear Models with High-Dimensional Binary Features by Parameter Confidence-Region Estimation.

Neural Comput 2020 10 14;32(10):1998-2031. Epub 2020 Aug 14.

RIKEN Center for Advanced Intelligent Project, Chuo-ku, Tokyo, 103-0027, Japan; Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi, 466-8555, Japan; and Center for Materials Research by Information Integration, National Institute for Material Science, Sengen, Tsukuba, Ibaraki, 305-0047, Japan

In this letter, we study an active learning problem for maximizing an unknown linear function with high-dimensional binary features. This problem is notoriously complex but arises in many important contexts. When the sampling budget, that is, the number of possible function evaluations, is smaller than the number of dimensions, it tends to be impossible to identify all of the optimal binary features. Therefore, in practice, only a small number of such features are considered, with the majority kept fixed at certain default values, which we call the . The main contribution of this letter is to formally study the working set heuristic and present a suite of theoretically robust algorithms for more efficient use of the sampling budget. Technically, we introduce a novel method for estimating the confidence regions of model parameters that is tailored to active learning with high-dimensional binary features. We provide a rigorous theoretical analysis of these algorithms and prove that a commonly used working set heuristic can identify optimal binary features with favorable sample complexity. We explore the performance of the proposed approach through numerical simulations and an application to a functional protein design problem.
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http://dx.doi.org/10.1162/neco_a_01310DOI Listing
October 2020

Unique Retinal Binding Pocket of Primate Blue-Sensitive Visual Pigment.

Biochemistry 2020 07 30;59(28):2602-2607. Epub 2020 Jun 30.

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

The visual pigments of humans contain 11- retinal as the chromophore of light perception, and its photoisomerization to the all- form initiates visual excitation in our eyes. It is well-known that three isomeric states of retinal (11-, all-, and 9-) are in photoequilibrium at very low temperatures such as 77 K. Here we report the lack of formation of the 9- form in monkey blue (MB) at 77 K, as revealed by light-induced difference Fourier transform infrared spectroscopy. This indicates that the chromophore binding pocket of MB does not accommodate the 9- form, even though it accommodates the all- form by twisting the chromophore. Mutation of the blue-specific tyrosine at position 265 to tryptophan, which is highly conserved in other animal rhodopsins, led to formation of the 9- form in MB, suggesting that Y265 is one of the determinants of the unique photochemistry in blue pigments. We also found that 9- retinal does not bind to MB opsin, implying that the chromophore binding pocket does not accommodate the 9- form at physiological temperature. The unique property of MB is discussed on the basis of the results presented here.
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http://dx.doi.org/10.1021/acs.biochem.0c00394DOI Listing
July 2020

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

Molecular Properties of New Enzyme Rhodopsins with Phosphodiesterase Activity.

ACS Omega 2020 May 27;5(18):10602-10609. Epub 2020 Apr 27.

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

The choanoflagellate contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme (Rh-PDE), which decreases the concentrations of cyclic nucleotides such as cGMP and cAMP in light, is a useful tool in optogenetics. Recently, eight additional Rh-PDE enzymes were found in choanoflagellate species, four from and the other four from other species. In this paper, we studied the molecular properties of these new Rh-PDEs, which were compared with Rh-PDE. Upon expression in HEK293 cells, four Rh-PDE proteins, including Rh-PDE2 and Rh-PDE3, exhibited no PDE activity when assessed by in-cell measurements and in vitro HPLC analysis. On the other hand, Rh-PDE1 showed light-dependent PDE activity toward cGMP, which absorbed maximally at 491 nm. Therefore, Rh-PDE1 is presumably responsible for colony inversion in by absorbing blue-green light. The molecular properties of Rh-PDE were similar to those of Rh-PDE, although the λ of Rh-PDE (516 nm) was considerably red-shifted from that of Rh-PDE (492 nm). One Rh-PDE, Rh-PDE, did not contain the retinal-binding Lys at TM7 and showed cAMP-specific PDE activity both in the dark and light. These results provide mechanistic insight into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.
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http://dx.doi.org/10.1021/acsomega.0c01113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227045PMC
May 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

Novel optogenetics tool: Gt_CCR4, a light-gated cation channel with high reactivity to weak light.

Biophys Rev 2020 Apr 12;12(2):453-459. Epub 2020 Mar 12.

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

Optogenetics is a growing technique which allows manipulation of biological events simply by illumination. The technique is appreciated especially in the neuroscience field because of its availability in controlling neuronal functions. A light-gated cation channel, Cr_ChR2 from Chlamydomonas reinhardtii, is the first and mostly applied to optogenetics for activating neuronal excitability. In addition, the molecular mechanism of Cr_ChR2 has been intensively studied by electrophysiology, spectroscopy, X-ray structural studies, etc. Novel cation channelrhodopsins from Guillardia theta, namely, Gt_CCR1-4, were discovered in 2016 and 2017. These channelrhodopsins are more homologous to haloarchaeal rhodopsins, particularly the proton pumps. Thus these cryptophyte-type light-gated cation channels are structurally and mechanistically distinct from chlorophyte channelrhodopsin such as Cr_ChR2. We here compared the photocurrent properties, cation selectivity, and kinetics between well-known Cr_ChR2 and Gt_CCR4. The light sensitivity of Gt_CCR4 is significantly higher than that of Cr_ChR2, while the channel open lifetime is in the same range as that of Cr_ChR2. Gt_CCR4 shows high Na selectivity in which the selectivity ratio for Na was 37-fold larger than that for Cr_ChR2, which primarily conducts H. On the other hand, Gt_CCR4 conducted almost no H and no Ca under physiological conditions. Other unique features and the applicability of Gt_CCR4 for optogenetics were discussed.
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http://dx.doi.org/10.1007/s12551-020-00676-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242533PMC
April 2020
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