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Development of an Outward Proton Pumping Rhodopsin with a New Record in Thermostability by Means of Amino Acid Mutations. 査読国際誌

Satoshi Yasuda, Tomoki Akiyama, Keiichi Kojima, Tetsuya Ueta, Tomohiko Hayashi, Satoshi Ogasawara, Satoru Nagatoishi, Kouhei Tsumoto, Naoki Kunishima, Yuki Sudo, Masahiro Kinoshita, Takeshi Murata

The Journal of Physical Chemistry B 126 ( 5 ) 1004 - 1015 2022年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

We have developed a methodology for identifying further thermostabilizing mutations for an intrinsically thermostable membrane protein. The methodology comprises the following steps: (1) identifying thermostabilizing single mutations (TSSMs) for residues in the transmembrane region using our physics-based method; (2) identifying TSSMs for residues in the extracellular and intracellular regions, which are in aqueous environment, using an empirical force field FoldX; and (3) combining the TSSMs identified in steps (1) and (2) to construct multiple mutations. The methodology is illustrated for thermophilic rhodopsin whose apparent midpoint temperature of thermal denaturation Tm is ∼91.8 °C. The TSSMs previously identified in step (1) were F90K, F90R, and Y91I with ΔTm ∼5.6, ∼5.5, and ∼2.9 °C, respectively, and those in step (2) were V79K, T114D, A115P, and A116E with ΔTm ∼2.7, ∼4.2, ∼2.6, and ∼2.3 °C, respectively (ΔTm denotes the increase in Tm). In this study, we construct triple and quadruple mutants, F90K+Y91I+T114D and F90K+Y91I+V79K+T114D. The values of ΔTm for these multiple mutants are ∼11.4 and ∼13.5 °C, respectively. Tm of the quadruple mutant (∼105.3 °C) establishes a new record in a class of outward proton pumping rhodopsins. It is higher than Tm of Rubrobacter xylanophilus rhodopsin (∼100.8 °C) that was the most thermostable in the class before this study.

DOI： 10.1021/acs.jpcb.1c08684

PubMed

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Proton-pumping rhodopsins in marine diatoms

Susumu Yoshizawa, Tomonori Azuma, Keiichi Kojima, Keisuke Inomura, Masumi Hasegawa, Yosuke Nishimura, Masuzu Kikuchi, Gabrielle Armin, Hideaki Miyashita, Kentaro Ifuku, Takashi Yamano, Adrian Marchetti, Hideya Fukuzawa, Yuki Sudo, Ryoma Kamikawa

bioRxiv 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cold Spring Harbor Laboratory

Abstract

Diatoms are a major phytoplankton group responsible for about 20% of Earth’s primary production. They carry out photosynthesis inside the plastid, an organelle obtained through eukaryote-eukaryote endosymbiosis. Recently, microbial rhodopsin, a photoreceptor distinct from chlorophyll-based photosystems, has been identified in certain diatoms. However, the physiological function of diatom rhodopsin is not well understood. Here we show that the diatom rhodopsin acts as a light-driven proton pump and localizes to the outermost membrane of the four membrane-bound complex plastids. Heterologous expression techniques were used to investigate the protein function and subcellular localization of diatom rhodopsin. Using model simulations, we further evaluated the physiological role of the acidic pool in the plastid produced by proton-transporting rhodopsin. Our results propose that the rhodopsin-derived acidic pool may be involved in a photosynthetic CO₂-concentrating mechanism and assist CO₂ fixation in diatom cells.

DOI： 10.1101/2022.01.18.476826

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Proton transfer pathway in anion channelrhodopsin-1 査読

Masaki Tsujimura, Keiichi Kojima, Shiho Kawanishi, Yuki Sudo, Hiroshi Ishikita

eLife 10 e72264 2021年12月

担当区分：責任著者掲載種別：研究論文（学術雑誌）出版者・発行元：eLife Sciences Publications, Ltd

Anion channelrhodopsin from <italic>Guillardia theta</italic> (<italic>Gt</italic>ACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant <italic>Gt</italic>ACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type <italic>Gt</italic>ACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

DOI： 10.7554/elife.72264

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その他リンク： https://cdn.elifesciences.org/articles/72264/elife-72264-v1.xml

Exploring the Retinal Binding Cavity of Archaerhodopsin-3 by Replacing the Retinal Chromophore With a Dimethyl Phenylated Derivative 査読

Taichi Tsuneishi, Masataka Takahashi, Masaki Tsujimura, Keiichi Kojima, Hiroshi Ishikita, Yasuo Takeuchi, Yuki Sudo

Frontiers in Molecular Biosciences 8 794948 2021年12月

担当区分：最終著者,　責任著者掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers Media SA

Rhodopsins act as photoreceptors with their chromophore retinal (vitamin-A aldehyde) and they regulate light-dependent biological functions. Archaerhodopsin-3 (AR3) is an outward proton pump that has been widely utilized as a tool for optogenetics, a method for controlling cellular activity by light. To characterize the retinal binding cavity of AR3, we synthesized a dimethyl phenylated retinal derivative, (2E,4E,6E,8E)-9-(2,6-Dimethylphenyl)-3,7-dimethylnona-2,4,6,8-tetraenal (DMP-retinal). QM/MM calculations suggested that DMP-retinal can be incorporated into the opsin of AR3 (archaeopsin-3, AO3). Thus, we introduced DMP-retinal into AO3 to obtain the non-natural holoprotein (AO3-DMP) and compared some molecular properties with those of AO3 with the natural A1-retinal (AO3-A1) or AR3. Light-induced pH change measurements revealed that AO3-DMP maintained slow outward proton pumping. Noteworthy, AO3-DMP had several significant changes in its molecular properties compared with AO3-A1 as follows; 1) spectroscopic measurements revealed that the absorption maximum was shifted from 556 to 508 nm and QM/MM calculations showed that the blue-shift was due to the significant increase in the HOMO-LUMO energy gap of the chromophore with the contribution of some residues around the chromophore, 2) time-resolved spectroscopic measurements revealed the photocycling rate was significantly decreased, and 3) kinetical spectroscopic measurements revealed the sensitivity of the chromophore binding Schiff base to attack by hydroxylamine was significantly increased. The QM/MM calculations show that a cavity space is present at the aromatic ring moiety in the AO3-DMP structure whereas it is absent at the corresponding <italic>β</italic>-ionone ring moiety in the AO3-A1 structure. We discuss these alterations of the difference in interaction between the natural A1-retinal and the DMP-retinal with binding cavity residues.

DOI： 10.3389/fmolb.2021.794948

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Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization 査読

Masuzu Kikuchi, Keiichi Kojima, Shin Nakao, Susumu Yoshizawa, Shiho Kawanishi, Atsushi Shibukawa, Takashi Kikukawa, Yuki Sudo

Scientific Reports 11 ( 1 ) 14756 2021年12月

担当区分：最終著者,　責任著者掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

<title>Abstract</title>Microbial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an <italic>Escherichia coli</italic> expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate <italic>Oxyrrhis marina</italic> (<italic>O. marina</italic> rhodopsin-2, <italic>Om</italic>R2) that can be expressed in <italic>E. coli</italic> cells. <italic>E. coli</italic> cells harboring the <italic>Om</italic>R2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified <italic>Om</italic>R2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-<italic>trans</italic> retinal chromophore, (2) the possession of the deprotonated counterion (p<italic>K</italic>_a = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of <italic>Om</italic>R2 expressed in <italic>E. coli</italic> cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

DOI： 10.1038/s41598-021-94181-w

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その他リンク： http://www.nature.com/articles/s41598-021-94181-w

An optogenetic assay method for electrogenic transporters using Escherichia coli co‐expressing light‐driven proton pump 査読

Masahiro Hayashi, Keiichi Kojima, Yuki Sudo, Atsuko Yamashita

Protein Science 30 ( 10 ) 2161 - 2169 2021年7月

Structure of a retinal chromophore of dark-adapted middle rhodopsin as studied by solid-state nuclear magnetic resonance spectroscopy 査読

Izuru Kawamura, Hayato Seki, Seiya Tajima, Yoshiteru Makino, Arisu Shigeta, Takashi Okitsu, Akimori Wada, Akira Naito, Yuki Sudo

Biophysics and Physicobiology 18 177 - 185 2021年

Lokiarchaeota archaeon schizorhodopsin-2 (LaSzR2) is an inward proton pump displaying a characteristic feature of acid-induced spectral blue-shift 査読

Keiichi Kojima, Susumu Yoshizawa, Masumi Hasegawa, Masaki Nakama, Marie Kurihara, Takashi Kikukawa, Yuki Sudo

Scientific Reports 10 ( 1 ) 20857 2020年11月

担当区分：最終著者,　責任著者掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

Abstract

The photoreactive protein rhodopsin is widespread in microorganisms and has a variety of photobiological functions. Recently, a novel phylogenetically distinctive group named ‘schizorhodopsin (SzR)’ has been identified as an inward proton pump. We performed functional and spectroscopic studies on an uncharacterised schizorhodopsin from the phylum Lokiarchaeota archaeon. The protein, LaSzR2, having an all-trans-retinal chromophore, showed inward proton pump activity with an absorption maximum at 549 nm. The pH titration experiments revealed that the protonated Schiff base of the retinal chromophore (Lys188, pK_a = 12.3) is stabilised by the deprotonated counterion (presumably Asp184, pK_a = 3.7). The flash-photolysis experiments revealed the presence of two photointermediates, K and M. A proton was released and uptaken from bulk solution upon the formation and decay of the M intermediate. During the M-decay, the Schiff base was reprotonated by the proton from a proton donating residue (presumably Asp172). These properties were compared with other inward (SzRs and xenorhodopsins, XeRs) and outward proton pumps. Notably, LaSzR2 showed acid-induced spectral ‘blue-shift’ due to the protonation of the counterion, whereas outward proton pumps showed opposite shifts (red-shifts). Thus, we can distinguish between inward and outward proton pumps by the direction of the acid-induced spectral shift.

DOI： 10.1038/s41598-020-77936-9

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その他リンク： https://www.nature.com/articles/s41598-020-77936-9

Mechanism of absorption wavelength shifts in anion channelrhodopsin-1 mutants 査読

Tsujimura M, Noji T, Saito K, Kojima K, Sudo Y, Ishikita H.

Biochim Biophys Acta (Bioenerg) 148349 - 148349 2020年11月

Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI 査読

Tetsuya Ueta, Keiichi Kojima, Tomoya Hino, Mikihiro Shibata, Shingo Nagano, Yuki Sudo

Biophysical Journal 119 ( 9 ) 1760 - 1770 2020年11月

Further thermo‐stabilization of thermophilic rhodopsin from Thermus thermophilus JL‐18 through engineering in extramembrane regions 査読

Tomoki Akiyama, Naoki Kunishima, Sayaka Nemoto, Kazuki Kazama, Masako Hirose, Yuki Sudo, Yoshinori Matsuura, Hisashi Naitow, Takeshi Murata

Proteins: Structure, Function, and Bioinformatics 89 ( 3 ) 301 - 310 2020年10月

掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Abstract

It is known that a hyperthermostable protein tolerable at temperatures over 100°C can be designed from a soluble globular protein by introducing mutations. To expand the applicability of this technology to membrane proteins, here we report a further thermo‐stabilization of the thermophilic rhodopsin from Thermus thermophilus JL‐18 as a model membrane protein. Ten single mutations in the extramembrane regions were designed based on a computational prediction of folding free‐energy differences upon mutation. Experimental characterizations using the UV‐visible spectroscopy and the differential scanning calorimetry revealed that four of ten mutations were thermo‐stabilizing: V79K, T114D, A115P, and A116E. The mutation‐structure relationship of the TR constructs was analyzed using molecular dynamics simulations at 300 K and at 1800 K that aimed simulating structures in the native and in the random‐coil states, respectively. The native‐state simulation exhibited an ion‐pair formation of the stabilizing V79K mutant as it was designed, and suggested a mutation‐induced structural change of the most stabilizing T114D mutant. On the other hand, the random‐coil‐state simulation revealed a higher structural fluctuation of the destabilizing mutant S8D when compared to the wild type, suggesting that the higher entropy in the random‐coil state deteriorated the thermal stability. The present thermo‐stabilization design in the extramembrane regions based on the free‐energy calculation and the subsequent evaluation by the molecular dynamics may be useful to improve the production of membrane proteins for structural studies.

DOI： 10.1002/prot.26015

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/prot.26015

A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage 査読

Masumi Hasegawa, Toshiaki Hosaka, Keiichi Kojima, Yosuke Nishimura, Yu Nakajima, Tomomi Kimura-Someya, Mikako Shirouzu, Yuki Sudo, Susumu Yoshizawa

Scientific Reports 10 ( 1 ) 16752 2020年10月

掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

Abstract

Microbial rhodopsin is a photoreceptor protein found in various bacteria and archaea, and it is considered to be a light-utilization device unique to heterotrophs. Recent studies have shown that several cyanobacterial genomes also include genes that encode rhodopsins, indicating that these auxiliary light-utilizing proteins may have evolved within photoautotroph lineages. To explore this possibility, we performed a large-scale genomic survey to clarify the distribution of rhodopsin and its phylogeny. Our surveys revealed a novel rhodopsin clade, cyanorhodopsin (CyR), that is unique to cyanobacteria. Genomic analysis revealed that rhodopsin genes show a habitat-biased distribution in cyanobacterial taxa, and that the CyR clade is composed exclusively of non-marine cyanobacterial strains. Functional analysis using a heterologous expression system revealed that CyRs function as light-driven outward H⁺ pumps. Examination of the photochemical properties and crystal structure (2.65 Å resolution) of a representative CyR protein, N2098R from Calothrix sp. NIES-2098, revealed that the structure of the protein is very similar to that of other rhodopsins such as bacteriorhodopsin, but that its retinal configuration and spectroscopic characteristics (absorption maximum and photocycle) are distinct from those of bacteriorhodopsin. These results suggest that the CyR clade proteins evolved together with chlorophyll-based photosynthesis systems and may have been optimized for the cyanobacterial environment.

DOI： 10.1038/s41598-020-73606-y

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その他リンク： https://www.nature.com/articles/s41598-020-73606-y

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation 査読

Han‐Min Ohn, Tamaki Mizuno, Yuki Sudo, Shin‐Ichi Miyoshi

Microbiology and Immunology 64 ( 9 ) 593 - 601 2020年8月

掲載種別：研究論文（学術雑誌）出版者・発行元：Wiley

Abstract

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L‐180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell‐associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.

DOI： 10.1111/1348-0421.12829

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/1348-0421.12829

Comparative Studies of the Fluorescence Properties of Microbial Rhodopsins: Spontaneous Emission Versus Photointermediate Fluorescence 査読

Keiichi Kojima, Rika Kurihara, Masayuki Sakamoto, Tsukasa Takanashi, Hikaru Kuramochi, Xiao Min Zhang, Haruhiko Bito, Tahei Tahara, Yuki Sudo

The Journal of Physical Chemistry B 124 ( 34 ) 7361 - 7367 2020年7月

Green-Sensitive, Long-Lived, Step-Functional Anion Channelrhodopsin-2 Variant as a High-Potential Neural Silencing Tool 査読

Keiichi Kojima, Natsuki Miyoshi, Atsushi Shibukawa, Srikanta Chowdhury, Masaki Tsujimura, Tomoyasu Noji, Hiroshi Ishikita, Akihiro Yamanaka, Yuki Sudo

The Journal of Physical Chemistry Letters 11 ( 15 ) 6214 - 6218 2020年7月

Methodology for Further Thermostabilization of an Intrinsically Thermostable Membrane Protein Using Amino Acid Mutations with Its Original Function Being Retained 査読

Satoshi Yasuda, Tomoki Akiyama, Sayaka Nemoto, Tomohiko Hayashi, Tetsuya Ueta, Keiichi Kojima, Takashi Tsukamoto, Satoru Nagatoishi, Kouhei Tsumoto, Yuki Sudo, Masahiro Kinoshita, Takeshi Murata

Journal of Chemical Information and Modeling 60 ( 3 ) 1709 - 1716 2020年3月

How Does a Microbial Rhodopsin RxR Realize Its Exceptionally High Thermostability with the Proton-Pumping Function Being Retained? 査読

Tomohiko Hayashi, Satoshi Yasuda, Kano Suzuki, Tomoki Akiyama, Kanae Kanehara, Keiichi Kojima, Mikio Tanabe, Ryuichi Kato, Toshiya Senda, Yuki Sudo, Takeshi Murata, Masahiro Kinoshita

The Journal of Physical Chemistry B 124 ( 6 ) 990 - 1000 2020年1月

Vectorial proton transport mechanism of RxR, a phylogenetically distinct and thermally stable microbial rhodopsin 査読国際誌

Keiichi Kojima, Tetsuya Ueta, Tomoyasu Noji, Keisuke Saito, Kanae Kanehara, Susumu Yoshizawa, Hiroshi Ishikita, Yuki Sudo

Scientific Reports 10 ( 1 ) 282 - 282 2020年1月

Bacterium Lacking a Known Gene for Retinal Biosynthesis Constructs Functional Rhodopsins 査読

Yu Nakajima, Keiichi Kojima, Yuichiro Kashiyama, Satoko Doi, Ryosuke Nakai, Yuki Sudo, Kazuhiro Kogure, Susumu Yoshizawa

Microbes and Environments 35 ( 4 ) ME20085 2020年

A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators 査読

David M. Needham, Susumu Yoshizawa, Toshiaki Hosaka, Camille Poirier, Chang Jae Choi, Elisabeth Hehenberger, Nicholas A. T. Irwin, Susanne Wilken, Cheuk-Man Yung, Charles Bachy, Rika Kurihara, Yu Nakajima, Keiichi Kojima, Tomomi Kimura-Someya, Guy Leonard, Rex R. Malmstrom, Daniel R. Mende, Daniel K. Olson, Yuki Sudo, Sebastian Sudek, Thomas A. Richards, Edward F. DeLong, Patrick J. Keeling, Alyson E. Santoro, Mikako Shirouzu, Wataru Iwasaki, Alexandra Z. Worden

Proceedings of the National Academy of Sciences 116 ( 41 ) 20574 - 20583 2019年9月

掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirR _DTS ) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirR _DTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae . Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

DOI： 10.1073/pnas.1907517116

PubMed

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その他リンク： https://pnas.org/doi/pdf/10.1073/pnas.1907517116

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