Publications by authors named "Yuji Furutani"

86 Publications

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

Optogenetic Modulation of Ion Channels by Photoreceptive Proteins.

Adv Exp Med Biol 2021 ;1293:73-88

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, Okazaki, Japan.

In these 15 years, researches to control cellular responses by light have flourished dramatically to establish "optogenetics" as a research field. In particular, light-dependent excitation/inhibition of neural cells using channelrhodopsins or other microbial rhodopsins is the most powerful and the most widely used optogenetic technique. New channelrhodopsin-based optogenetic tools having favorable characteristics have been identified from a wide variety of organisms or created through mutagenesis. Despite the great efforts, some neuronal activities are still hard to be manipulated by the channelrhodopsin-based tools, indicating that complementary approaches are needed to make optogenetics more comprehensive. One of the feasible and complementary approaches is optical control of ion channels using photoreceptive proteins other than channelrhodopsins. In particular, animal opsins can modulate various ion channels via light-dependent G protein activation. In this chapter, we summarize how such alternative optogenetic tools work and they will be improved.
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http://dx.doi.org/10.1007/978-981-15-8763-4_5DOI Listing
February 2021

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

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

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

Regulation of Photocycle Kinetics of Photoactive Yellow Protein by Modulating Flexibility of the β-Turn.

J Phys Chem B 2020 02 17;124(8):1452-1459. Epub 2020 Feb 17.

Graduate School of Materials Science , Nara Institute of Science and Technology , Ikoma , Nara 630-0192 , Japan.

The role of the significant flexibility of the β-turn in photoactive yellow protein (PYP) due to Gly115 was studied. G115A and G115P mutations were observed to accelerate the photocycle and shift the equilibrium between the late photocycle intermediate (pB) and its precursor (pR) toward pR. Thermodynamic analysis of dark-state recovery from pB demonstrated that the transition state (pB) has a negative change in transition heat capacity, suggesting that an exposed hydrophobic surface of pB is buried in pB. Fourier transform infrared spectroscopy showed that the structural ensemble of pB is populated by the compact structure in G115P. Taken together, the rigid structure induced by mutation of Gly115 facilitates its transition to pB, which adopts a substantially more compact structure as opposed to the ensemble-averaged structure of pB. The photocycle kinetics of PYP may be fine-tuned by modulating the flexibility of the 115 loop to activate an appropriate number of transducer proteins.
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http://dx.doi.org/10.1021/acs.jpcb.9b11879DOI Listing
February 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

Vibrational and Molecular Properties of Mg Binding and Ion Selectivity in the Magnesium Channel MgtE.

J Phys Chem B 2018 10 10;122(42):9681-9696. Epub 2018 Oct 10.

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science , National Institutes of Natural Sciences , 38 Nishigo-Naka , Myodaiji, Okazaki 444-8585 , Japan.

Magnesium ions (Mg) are crucial for various biological processes. A bacterial Mg channel, MgtE, tightly regulates the intracellular Mg concentration. Previous X-ray crystal structures showed that MgtE forms a dimeric structure composed of a total of 10 transmembrane α helices forming a central pore, and intracellular soluble domains constituting a Mg sensor. The ion selectivity for Mg over Ca resides at a central cavity in the transmembrane pore of MgtE, involving a conserved aspartate residue (Asp432) from each monomer. Here, we applied ion-exchange-induced difference FTIR spectroscopy to analyze the interactions between MgtE and divalent cations, Mg and Ca. Using site-directed mutagenesis, vibrational bands at 1421 (Mg), 1407 (Mg), ∼1440 (Ca), and 1390 (Ca) cm were assigned to symmetric carboxylate stretching modes of Asp432, involved in the ion coordination. Conservative modifications of the central cavity by Asp432Glu or Ala417Leu mutations resulted in the disappearance of the Mg-sensitive carboxylate bands, suggesting a highly optimized geometry for accommodating a Mg ion. The dependency of the vibrational changes on Mg and Ca concentrations revealed the presence of a two different classes of binding sites: a high affinity site for Mg ( K ≈ 0.3 mM) with low Ca affinity ( K ≈ 80 mM), and a medium affinity site for Mg ( K ≈ 2 mM) and Ca ( K ≈ 6 mM), tentatively assigned to the central cavity and the sensor domain, respectively. With the aid of molecular dynamics simulation and normal-mode analysis by quantum chemistry, we confirm that changes in carboxylate bands of the high affinity binding site originate from Asp432 in the central cavity.
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http://dx.doi.org/10.1021/acs.jpcb.8b07967DOI Listing
October 2018

Structural properties determining low K affinity of the selectivity filter in the TWIK1 K channel.

J Biol Chem 2018 05 15;293(18):6969-6984. Epub 2018 Mar 15.

From the Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, and

Canonical K channels are tetrameric and highly K-selective, whereas two-pore-domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore-domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange-induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1's surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful "molecular fingerprints" for assessing the properties of other K channels.
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http://dx.doi.org/10.1074/jbc.RA118.001817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936812PMC
May 2018

"In situ" observation of the role of chloride ion binding to monkey green sensitive visual pigment by ATR-FTIR spectroscopy.

Phys Chem Chem Phys 2018 Jan;20(5):3381-3387

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

Long-wavelength-sensitive (LWS) pigment possesses a chloride binding site in its protein moiety. The binding of chloride alters the absorption spectra of LWS; this is known as the chloride effect. Although the two amino acid substitutions of His197 and Lys200 influence the chloride effect, the molecular mechanism of chloride binding, which underlies the spectral tuning, has yet to be clarified. In this study, we applied ATR-FTIR spectroscopy to monkey green (MG) pigment to gain structural information of the chloride binding site. The results suggest that chloride binding stabilizes the β-sheet structure on the extracellular side loop with perturbation of the retinal polyene chain, promotes a hydrogen bonding exchange with the hydroxyl group of Tyr, and alters the protonation state of carboxylate. Combining with the results of the binding analyses of various anions (Br, I and NO), our findings suggest that the anion binding pocket is organized for only Cl (or Br) to stabilize conformation around the retinal chromophore, which is functionally relevant with absorbing long wavelength light.
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http://dx.doi.org/10.1039/c7cp07277eDOI Listing
January 2018

Ion-protein interactions of a potassium ion channel studied by attenuated total reflection Fourier transform infrared spectroscopy.

Authors:
Yuji Furutani

Biophys Rev 2018 Apr 22;10(2):235-239. Epub 2017 Nov 22.

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki, 444-8585, Japan.

An understanding of ion-protein interactions is key to a better understanding of the molecular mechanisms of proteins, such as enzymes, ion channels, and ion pumps. A potassium ion channel, KcsA, has been extensively studied in terms of ion selectivity. Alkali metal cations in the selectivity filter were visualized by X-ray crystallography. Infrared spectroscopy has an intrinsically higher structural sensitivity due to frequency changes in molecular vibrations interacting with different ions. In this review article, I attempt to summarize ion-exchange-induced differences in Fourier transform infrared spectroscopy, as applied to KcsA, to explain how this method can be utilized to study ion-protein interactions in the KcsA selectivity filter. A band at 1680 cm in the amide I region would be a marker band for the ion occupancy of K, Rb, and Cs in the filter. The band at 1627 cm observed in both Na and Li conditions suggests that the selectivity filter similarly interacts with these ions. In addition to the structural information, the results show that the titration of K ions provides quantitative information on the ion affinity of the selectivity filter.
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http://dx.doi.org/10.1007/s12551-017-0337-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899698PMC
April 2018

The Impact of the Polymer Chain Length on the Catalytic Activity of Poly(N-vinyl-2-pyrrolidone)-supported Gold Nanoclusters.

Sci Rep 2017 08 29;7(1):9579. Epub 2017 Aug 29.

Division of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.

Poly(N-vinyl-2-pyrrolidone) (PVP) of varying molecular weight (M  = 40-360 kDa) were employed to stabilize gold nanoclusters of varying size. The resulting Au:PVP clusters were subsequently used as catalysts for a kinetic study on the sized-dependent aerobic oxidation of 1-indanol, which was monitored by time-resolved in situ infrared spectroscopy. The obtained results suggest that the catalytic behaviour is intimately correlated to the size of the clusters, which in turn depends on the molecular weight of the PVPs. The highest catalytic activity was observed for clusters with a core size of ~7 nm, and the size of the cluster should increase with the molecular weight of the polymer in order to maintain optimal catalytic activity. Studies on the electronic and colloid structure of these clusters revealed that the negative charge density on the cluster surface also strongly depends on the molecular weight of the stabilizing polymers.
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http://dx.doi.org/10.1038/s41598-017-10165-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575105PMC
August 2017

A ciliary opsin in the brain of a marine annelid zooplankton is ultraviolet-sensitive, and the sensitivity is tuned by a single amino acid residue.

J Biol Chem 2017 08 16;292(31):12971-12980. Epub 2017 Jun 16.

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, Okazaki 444-8585, Japan; Department of Structural Molecular Science, Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193, Japan.

Ciliary opsins were classically thought to function only in vertebrates for vision, but they have also been identified recently in invertebrates for non-visual photoreception. Larvae of the annelid are used as a zooplankton model, and this zooplankton species possesses a "vertebrate-type" ciliary opsin (named c-opsin) in the brain. c-opsin is suggested to relay light signals for melatonin production and circadian behaviors. Thus, the spectral and biochemical characteristics of this c-opsin would be directly related to non-visual photoreception in this zooplankton model. Here we demonstrate that the c-opsin can sense UV to activate intracellular signaling cascades and that it can directly bind exogenous all--retinal. These results suggest that this c-opsin regulates circadian signaling in a UV-dependent manner and that it does not require a supply of 11--retinal for photoreception. Avoidance of damaging UV irradiation is a major cause of large-scale daily zooplankton movement, and the observed capability of the c-opsin to transmit UV signals and bind all-retinal is ideally suited for sensing UV radiation in the brain, which presumably lacks enzymes producing 11--retinal. Mutagenesis analyses indicated that a unique amino acid residue (Lys-94) is responsible for c-opsin-mediated UV sensing in the brain. We therefore propose that acquisition of the lysine residue in the c-opsin would be a critical event in the evolution of to enable detection of ambient UV light. In summary, our findings indicate that the c-opsin possesses spectral and biochemical properties suitable for UV sensing by the zooplankton model.
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http://dx.doi.org/10.1074/jbc.M117.793539DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546036PMC
August 2017

Structural insights into the nucleotide base specificity of P2X receptors.

Sci Rep 2017 03 23;7:45208. Epub 2017 Mar 23.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors.
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http://dx.doi.org/10.1038/srep45208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362899PMC
March 2017

Live-cell single-molecule imaging of the cytokine receptor MPL for analysis of dynamic dimerization.

J Mol Cell Biol 2016 12 9;8(6):553-555. Epub 2016 Jun 9.

Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 1130033, Japan

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http://dx.doi.org/10.1093/jmcb/mjw027DOI Listing
December 2016

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins.

J Biol Chem 2015 Nov 28;290(45):27176-27187. Epub 2015 Sep 28.

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, Okazaki, 444-8585, Japan,; Department of Structural Molecular Science, SOKENDAI (The Graduate University for Advanced Studies), Hayama, Kanagawa 240-0193, Japan.

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.
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http://dx.doi.org/10.1074/jbc.M115.666305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646394PMC
November 2015

Chimeras of channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii exhibit distinctive light-induced structural changes from channelrhodopsin-2.

J Biol Chem 2015 May 21;290(18):11623-34. Epub 2015 Mar 21.

From the Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan, PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, Department of Structural Molecular Science, Graduate University for Advanced Studies (SOKENDAI), 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan,

Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.
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http://dx.doi.org/10.1074/jbc.M115.642256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416865PMC
May 2015

Attenuated total reflectance spectroscopy with chirped-pulse upconversion.

Opt Express 2014 Dec;22(24):29611-6

Chirped-pulse upconversion technique has been applied to attenuated total reflectance (ATR) infrared spectroscopy. An extremely broadband infrared pulse was sent to an ATR diamond prism and the reflected pulse was converted to the visible by using four-wave mixing in krypton gas. Absorption spectra of liquids in the range from 200 to 5500 cm(-1) were measured with a visible spectrometer on a single-shot basis. The system was applied to observe the dynamics of exchanging process of two solvents, water and acetone, which give clear vibrational spectral contrast. We observed that the exchange was finished within ∼ 10 ms.
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http://dx.doi.org/10.1364/OE.22.029611DOI Listing
December 2014

Specific interactions between alkali metal cations and the KcsA channel studied using ATR-FTIR spectroscopy.

Biophys Physicobiol 2015 12;12:37-45. Epub 2015 Sep 12.

Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Aichi 466-8555, Japan.

The X-ray structure of KcsA, a eubacterial potassium channel, displays a selectivity filter composed of four parallel peptide strands. The backbone carbonyl oxygen atoms of these strands solvate multiple K(+) ions. KcsA structures show different distributions of ions within the selectivity filter in solutions containing different cations. To assess the interactions of cations with the selectivity filter, we used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. Ion-exchange-induced ATR-FTIR difference spectra were obtained by subtracting the spectrum of KcsA soaked in K(+) solution from that obtained in Li(+), Na(+), Rb(+), and Cs(+) solutions. Large spectral changes in the amide-I and -II regions were observed upon replacing K(+) with smaller-sized cations Li(+) and Na(+) but not with larger-sized cations Rb(+) and Cs(+). These results strongly suggest that the selectivity filter carbonyls coordinating Rb(+) or Cs(+) adopt a conformation similar to those coordinating K(+) (cage configuration), but those coordinating Li(+) or Na(+) adopt a conformation (plane configuration) considerably different from those coordinating K(+). We have identified a cation-type sensitive amide-I band at 1681 cm(-1) and an insensitive amide-I band at 1659 cm(-1). The bands at 1650, 1639, and 1627 cm(-1) observed for Na(+)-coordinating carbonyls were almost identical to those observed in Li(+) solution, suggesting that KcsA forms a similar filter structure in Li(+) and Na(+) solutions. Thus, we conclude that the filter structure adopts a collapsed conformation in Li(+) solution that is similar to that in Na(+) solution but is in clear contrast to the X-ray crystal structure of KcsA with Li(+).
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http://dx.doi.org/10.2142/biophysico.12.0_37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736833PMC
August 2016

Distribution of mammalian-like melanopsin in cyclostome retinas exhibiting a different extent of visual functions.

PLoS One 2014 24;9(9):e108209. Epub 2014 Sep 24.

Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Osaka, Japan.

Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108209PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177573PMC
June 2015

His166 is the Schiff base proton acceptor in attractant phototaxis receptor sensory rhodopsin I.

Biochemistry 2014 Sep 8;53(37):5923-9. Epub 2014 Sep 8.

Department of Frontier Materials, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.

Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore's Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein's normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X-H stretch modes in light-minus-dark difference spectra; their assignment with (15)N labeling and site-directed mutagenesis demonstrates that His166 is the SB proton acceptor during the photochemical reaction cycle of the wild-type SRI-HtrI complex.
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http://dx.doi.org/10.1021/bi500831nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172204PMC
September 2014

Infrared spectroscopic studies on the V-ATPase.

Biochim Biophys Acta 2015 Jan 8;1847(1):134-41. Epub 2014 Aug 8.

PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan; Department of Chemistry, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.

V-ATPase is an ATP-driven rotary motor that vectorially transports ions. Together with F-ATPase, a homologous protein, several models on the ion transport have been proposed, but their molecular mechanisms are yet unknown. V-ATPase from Enterococcus hirae forms a large supramolecular protein complex (total molecular weight: ~700,000) and physiologically transports Na⁺ and Li⁺ across a hydrophobic lipid bilayer. Stabilization of these cations in the binding site has been discussed on the basis of X-ray crystal structures of a membrane-embedded domain, the K-ring (Na⁺ and Li⁺ bound forms). Sodium or lithium ion binding-induced difference FTIR spectra of the intact E. hirae V-ATPase have been measured in aqueous solution at physiological temperature. The results suggest that sodium or lithium ion binding induces the deprotonation of Glu139, a hydrogen-bonding change in the tyrosine residue and rigid α-helical structures. Identical difference FTIR spectra between the entire V-ATPase complex and K-ring strongly suggest that protein interaction with the I subunit does not cause large structural changes in the K-ring. This result supports the previously proposed Na⁺ transport mechanism by V-ATPase stating that a flip-flop movement of a carboxylate group of Glu139 without large conformational changes in the K-ring accelerates the replacement of a Na⁺ ion in the binding site. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.
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http://dx.doi.org/10.1016/j.bbabio.2014.07.020DOI Listing
January 2015

Self-assembly of the chaperonin GroEL nanocage induced at submicellar detergent.

Sci Rep 2014 Jul 8;4:5614. Epub 2014 Jul 8.

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Protein nanoassemblies possess unique advantage in biomedical applications such as drug delivery, biocatalysis and vaccine development. Despite recent accomplishment in atomic structure data, the underlying molecular mechanism of protein self-assembly remains elusive, where considerable heterogeneity is often involved. Here we use E. coli chaperonin GroEL, a tetradecameric protein with a molecular weight of 805 kDa, to probe its transformation from cage-like oligomers to protein nanofibers. We show that sodium dodecyl sulfate (SDS), a widely-used protein denaturant, at submicellar concentration binds to and causes partial distortion of GroEL apical domain. Subsequently, the GroEL apical domain with altered secondary structural content converts the GroEL oligomers into modular structural units which are observed to self-assemble into cylindrical nanofibers under an agitated incubation in a physiological buffer. Interestingly, through targeted mutagenesis where two cysteine residues are introduced at the entry site of GroEL cage, we found that the formation of GroEL nanoassembly could be modulated depending on the redox condition of incubation. Without the need of chemical engineering, tunable GroEL nanofibers built by controlled-assembly are among the largest nanoscale bioassembly with broad applications.
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http://dx.doi.org/10.1038/srep05614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4085630PMC
July 2014

Hydrogen-bonding changes of internal water molecules upon the actions of microbial rhodopsins studied by FTIR spectroscopy.

Biochim Biophys Acta 2014 May 13;1837(5):598-605. Epub 2013 Sep 13.

Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan. Electronic address:

Microbial rhodopsins are classified into type-I rhodopsins, which utilize light energy to perform wide varieties of function, such as proton pumping, ion pumping, light sensing, cation channels, and so on. The crystal structures of several type-I rhodopsins were solved and the molecular mechanisms have been investigated based on the atomic structures. However, the crystal structures of proteins of interest are not always available and the basic architectures are sometimes quite similar, which obscures how the proteins achieve different functions. Stimulus-induced difference FTIR spectroscopy is a powerful tool to detect minute structural changes providing a clue for elucidating the molecular mechanisms. In this review, the studies on type-I rhodopsins from fungi and marine bacteria, whose crystal structures have not been solved yet, were summarized. Neurospora rhodopsin and Leptosphaeria rhodopsin found from Fungi have sequence similarity. The former has no proton pumping function, while the latter has. Proteorhodopsin is another example, whose proton pumping machinery is altered at alkaline and acidic conditions. We described how the structural changes of protein were different and how water molecules were involved in them. We reviewed the results on dynamics of the internal water molecules in pharaonis halorhodopsin as well. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.
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http://dx.doi.org/10.1016/j.bbabio.2013.09.004DOI Listing
May 2014

Large spectral change due to amide modes of a β-sheet upon the formation of an early photointermediate of middle rhodopsin.

J Phys Chem B 2013 Apr 26;117(13):3449-58. Epub 2013 Mar 26.

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigo-Naka, Myodaiji, Okazaki 444-8585, Japan.

Rhodopsin contains retinal as the chromophore within seven transmembrane helices. Recently, we found a unique rhodopsin (middle rhodopsin, MR), which is evolutionarily located between the well-studied bacteriorhodopsin and sensory rhodopsin II, and which accommodates three retinal isomers in its ground state (the all-trans, the 13-cis, and, uniquely, the 11-cis isomers). In this study, we investigated structural changes of both the protein moiety and the retinal chromophore during photocycles of MR by time-resolved Fourier-transform infrared spectroscopy. Three photointermediates with decay time constants of 95 μs, 0.9 ms, and >~10 ms were identified by the global exponential fitting analysis. The first and third intermediates were attributed to the all-trans photocycle, in accordance with recently published results, whereas the second intermediate was likely one that was spectroscopically silent in the visible region and that was formed between the first and third states or resulted from the activation of the 13-cis isomer. By comparing light-induced difference spectra with various isotope labels in either the retinal or the protein moiety, we concluded that a β-sheet structure in the hydrophilic part was significantly altered during the all-trans photocycle of MR, which may involve an active state of the protein. This feature is characteristic of MR among microbial (type-1) rhodopsins.
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http://dx.doi.org/10.1021/jp308765tDOI Listing
April 2013

Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode.

Biophysics (Nagoya-shi) 2013 10;9:123-9. Epub 2013 Aug 10.

UNISOKU Co., Ltd., 2-4-3 Kasugano, Hirakata, Osaka 573-0131, Japan.

Attenuated total reflectance (ATR)-FTIR spectroscopy has been widely used to probe protein structural changes under various stimuli, such as light absorption, voltage change, and ligand binding, in aqueous conditions. Time-resolved measurements require a trigger, which can be controlled electronically; therefore, light and voltage changes are suitable. Here we developed a novel, rapid buffer-exchange system for time-resolved ATR-FTIR spectroscopy to monitor the ligand- or ion-binding re-action of a protein. By using the step-scan mode (time resolution; 2.5 ms), we confirmed the completion of the buffer-exchange reaction within ∼25 ms; the process was monitored by the infrared absorption change of a nitrate band at 1,350 cm(-1). We also demonstrated the anion-binding reaction of a membrane protein, Natronomonas pharaonis halorhodopsin (pHR), which binds a chloride ion in the initial anion-binding site near the retinal chromophore. The formation of chloride- or nitrate-bound pHR was confirmed by an increase of the retinal absorption band at 1,528 cm(-1). It also should be noted that low sample consumption (∼1 µg of protein) makes this new method a powerful technique to understand ligand-protein and ion-protein interactions, particularly for membrane proteins.
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http://dx.doi.org/10.2142/biophysics.9.123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4629687PMC
August 2016

ATR-FTIR Spectroscopy Revealing the Different Vibrational Modes of the Selectivity Filter Interacting with K(+) and Na(+) in the Open and Collapsed Conformations of the KcsA Potassium Channel.

J Phys Chem Lett 2012 Dec 10;3(24):3806-10. Epub 2012 Dec 10.

†Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.

The potassium channel is highly selective for K(+) over Na(+), and the selectivity filter binds multiple dehydrated K(+) ions upon permeation. Here, we applied attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to extract ion-binding-induced signals of the KcsA potassium channel at neutral pH. Shifts in the peak of the amide-I signal towards lower vibrational frequencies were observed as K(+) was replaced with Na(+). These ion species-specific shifts deduced the selectivity filter as the source of the signal, which was supported by the spectra of a mutant for the selectivity filter (Y78F). The difference FTIR spectra between the solution containing various concentrations of K(+) and that containing pure Na(+) demonstrated two types of peak shifts of the amide-I vibration in response to the K(+) concentration. These signals represent the binding of K(+) ions to the different sites in the selectivity filter with different dissociation constants (KD = 9 or 18 mM).
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http://dx.doi.org/10.1021/jz301721fDOI Listing
December 2012

Dynamics of Dangling Bonds of Water Molecules in pharaonis Halorhodopsin during Chloride Ion Transportation.

J Phys Chem Lett 2012 Oct 28;3(20):2964-9. Epub 2012 Sep 28.

#Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.

Ion transportation via the chloride ion pump protein pharaonis halorhodopsin (pHR) occurs through the sequential formation of several intermediates during a photocyclic reaction. Although the structural details of each intermediate state have been studied, the role of water molecules in the translocation of chloride ions inside of the protein at physiological temperatures remains unclear. To analyze the structural dynamics of water inside of the protein, we performed time-resolved Fourier transform infrared (FTIR) spectroscopy under H2O or H2(18)O hydration and successfully assigned water O-H stretching bands. We found that a dangling water band at 3626 cm(-1) in pHR disappears in the L1 and L2 states. On the other hand, relatively intense positive bands at 3605 and 3608 cm(-1) emerged upon the formation of the X(N) and O states, respectively, suggesting that the chloride transportation is accompanied by dynamic rearrangement of the hydrogen-bonding network of the internal water molecules in pHR.
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http://dx.doi.org/10.1021/jz301287nDOI Listing
October 2012

Comparative FTIR study of a new fungal rhodopsin.

J Phys Chem B 2012 Oct 25;116(39):11881-9. Epub 2012 Sep 25.

Department of Frontier Materials, Nagoya Institute of Technology, Nagoya 466-8555, Japan.

Bacteriorhodopsin (BR) is a light-driven proton pump of halophilic Archaea , and BR-like proton-pumping rhodopsins have been discovered in Bacteria and Eucarya as well. Leptosphaeria rhodopsin (LR) and Phaeosphaeria Rhodopsin 2 (PhaeoRD2) are both fungal rhodopsins in such a functional class, even though they belong to different branches of the phylogenetic tree. In this study, we compared light-induced structural changes in the K, L, and M photointermediates for PhaeoRD2, LR, and BR using low-temperature Fourier transform infrared (FTIR) spectroscopy. We observed a strongly hydrogen-bonded water molecule in PhaeoRD2 (water O-D stretch in D(2)O at 2258 cm(-1)) as well as in LR and BR. This observation provided additional experimental evidence to the concept that strongly hydrogen-bonded water molecule is the functional determinant of light-driven proton pumping. The difference FTIR spectra for all the K, L, and M states are surprisingly similar between PhaeoRD2 and LR, but not for BR. PhaeoRD2 is more homologous to LR than to BR, but the difference is small. The amino acid identities between PhaeoRD2 and LR, and between PhaeoRD2 and BR are 34.5% and 30.2%, respectively. In addition, the amino acids uniquely identical for the fungal rhodopsins are located rather far from the retinal chromophore. In fact, the amino acid identities within 4 Å from retinal are the same among PhaeoRD2, LR, and BR. For more than 100 amino acids located within 12 Å from retinal, the identities are 48.7% between PhaeoRD2 and LR, 46.0% between PhaeoRD2 and BR, and 47.8% between LR and BR. These results suggest that protein core structures are equally different among the three rhodopsins. Thus, the identical FTIR spectra between PhaeoRD2 and LR (but not BR), even for the K state, indicate that fungal rhodopsins possess some common structural motif and dynamics not obvious from the amino acid sequences.
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http://dx.doi.org/10.1021/jp306993aDOI Listing
October 2012

Protein-bound water molecules in primate red- and green-sensitive visual pigments.

Biochemistry 2012 Feb 2;51(6):1126-33. Epub 2012 Feb 2.

Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.

Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D(2)O and D(2)(18)O in the whole midinfrared region. The observed water vibrations are 6-8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-cis and 9-cis forms of rhodopsin, but different vibrational bands are observed at >2550 cm(-1) for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm(-1) for MR and 2308 cm(-1) for MG) are observed for the all-trans form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl(-)-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O-D stretching vibrations for MR and MG are lower as the λ(max) is red-shifted, suggesting that water molecules are involved in the color tuning of our vision.
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http://dx.doi.org/10.1021/bi201676yDOI Listing
February 2012
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