記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bpj.2017.11.004

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0177907

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER JAPAN KK  

3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant.

DOI： 10.1007/s00792-016-0811-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MICROBIOLOGY SOC  

Ribosome dimers are a translationally inactive form of ribosomes found in Escherichia coli and many other bacterial cells. In this study, we found that the 70S ribosomes of Bacillus subtilis dimerized during the early stationary phase and these dimers remained in the cytoplasm until regrowth was initiated. Ribosome dimerization during the stationary phase required the hpf gene, which encodes a homologue of the E. coli hibernation-promoting factor (Hpf). The expression of hpf was induced at an early stationary phase and its expression was observed throughout the rest of the experimental period, including the entire 6 h of the stationary phase. Ribosome dimerization followed the induction of hpf in WT cells, but the dimerization was impaired in cells harbouring a deletion in the hpf gene. Although the absence of ribosome dimerization in these Hpf-deficient cells did not affect their viability in the stationary phase, their ability to regrow from the stationary phase decreased. Thus, following the transfer of stationary-phase cells to fresh LB medium, Δhpf mutant cells grew slower than WT cells. This observed lag in growth of Δhpf cells was probably due to a delay in restoring their translational activity. During regrowth, the abundance of ribosome dimers in WT cells decreased with a concomitant increase in the abundance of 70S ribosomes and growth rate. These results suggest that the ribosome dimers, by providing 70S ribosomes to the cells, play an important role in facilitating rapid and efficient regrowth of cells under nutrient-rich conditions.

DOI： 10.1099/mic.0.000234

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担当区分：筆頭著者,　最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier Inc.  

Specific ATP binding to the ε subunit of thermophilic F1-ATPase has been utilized for the biosensors of
ATP in vivo. I report here that the ε subunit containing R103A/R115A mutations can bind ATP with a
dissociation constant at 52 nM, which is two orders of magnitude higher affinity than the wild type. The
mutant retained specificity for ATP; ADP and GTP bound to the mutant with dissociation constants 16
and 53 muM, respectively. Thus, the mutant would be a good platform for various types of nucleotide
biosensor with appropriate modifications.

DOI： 10.1016/j.bbrc.2015.12.121

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.

DOI： 10.1080/09168451.2014.890033

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

F1-ATPase from Bacillus subtilis (BF1) is severely suppressed by the MgADP inhibition. Here, we have tested if this is due to the loss of nucleotide binding to the noncatalytic site that is required for the activation. Measurements with a tryptophan mutant of BF1 indicated that the noncatalytic sites could bind ATP normally. Furthermore, the mutant BF1 that cannot bind ATP to the noncatalytic sites showed much lower ATPase activity. It was concluded that the cause of strong MgADP inhibition of BF1 is not the weak nucleotide binding to the noncatalytic sites but the other steps required for the activation.

DOI： 10.1371/journal.pone.0107197

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Public Library of Science  

DOI： 10.1371/journal.pone.0073888

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：[極限環境生物学会]学会事務局  

CiNii Article

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PORTLAND PRESS LTD  

ATP binding to the ϵ subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ϵ subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ϵ subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ϵ subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ϵ subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ϵ subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ϵ subunit was in its extended-state conformation. The present study reveals a novel role of the ϵ subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.

DOI： 10.1042/BJ20110443

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：CELL PRESS  

Subunit epsilon of bacterial and chloroplast F(O)F(1)-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit epsilon can adopt two conformations. In the "extended", inhibitory conformation, its two C-terminal alpha-helices are stretched along subunit gamma. In the "contracted", noninhibitory conformation, these helices form a hairpin. The transition of subunit epsilon from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59 degrees C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit epsilon and in the N-terminus of subunit gamma was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 microM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit beta were found to stabilize the extended conformation of epsilon. Binding of ATP directly to epsilon was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 microM) suggests that subunit epsilon probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value.

DOI： 10.1016/j.bpj.2009.10.023

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The F1-ATPase, the soluble part of FoF1-ATP synthase, is a rotary molecular motor consisting of α3β3γδε. The γ and ε subunits rotate relative to the α3β3δ sub-complex on ATP hydrolysis by the β subunit. The ε subunit is known as an endogenous inhibitor of the ATPase activity of the F1-ATPase and is believed to function as a regulator of the ATP synthase. This inhibition by the ε subunit (ε inhibition) of F1-ATPase from thermophilic Bacillus PS3 was analyzed by single molecule measurements. By using a mutant ε subunit deficient in ATP binding, reversible transitions between active and inactive states were observed. Analysis of pause and rotation durations showed that the ε inhibition takes a different path from the ADP-Mg inhibition. Furthermore, the addition of the mutant ε subunit to the α3β3γ sub-complex was found to facilitate recovery of the ATPase activity from the ADP-Mg inhibition. Thus, it was concluded that these two inhibitions are essentially exclusive of each other.

DOI： 10.2142/biophysics.6.59

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Effect of epsilon subunit on the nucleotide binding to the catalytic sites of F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)) has been tested by using alpha(3)beta(3)gamma and alpha(3)beta(3)gammaepsilon complexes of TF(1) containing betaTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the epsilon subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the epsilon subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.

DOI： 10.1016/j.bbrc.2009.09.092

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATL ACAD SCIENCES  

Adenosine 5'-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the epsilon subunit of the bacterial F(o)F(1)-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 muM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of F(o)F(1)-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

DOI： 10.1073/pnas.0904764106

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

PubMed

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.bbrc.2006.09.001

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担当区分：筆頭著者,　最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.febslet.2005.11.036

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M506160200

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.

DOI： 10.1038/nature03277

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.45.S244_4

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.45.S209_1

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：SPRINGER  

F-1 enzyme is known as the world smallest rotary motor, but its real biological function, namely the synthesis of ATP, has remained elusive. We describe the use of polydimethylsiloxane (PDMS) micrometer chamber arrays for the single molecule study of the efficiency of this molecular motor/generator. The patterned PDMS device allowed the enclosing of extremely small volumes of water solution. This packing was stable and tight. In this unprecedently small volumes (fL), activity of a unique enzyme results in rapid detectable (mu M) changes in the concentration of its substrates and products. We employed this technique to follow directly for the first time the ATP consumption of F-1 as it produces mechanical work. As expected, the enzyme was found to function with a yield close to 100%. We then turned to the ATP-generating role of F1. We attached a magnetic bead to the protein and used magnetic tweezers to force backward rotations of the enzyme. In the presence of ADP and Pi, we could detect the synthesis of ATP upon mechanical rotation at 10 Hz. As all the system was trapped in a very small volumes this ATP could not diffuse and F-1 itself served as a probe to measure the amount of produced ATP. The mechanochemical coupling was found very tight.

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：ROYAL SOC CHEMISTRY  

Planar bilayer membranes have been widely used for electrophysiological analysis of membrane proteins in laboratories. We previously reported that planar lipid bilayer can be reconstituted at 100 similar to 150 mu m tapered hole fabricated in a silicon-based micro-fluidic channel system. However, for the measurement of ion channel current, which requires pico-ampere sensitivity, the substrate material should be non-conductive material to eliminate the electric noise. Thus, we fabricated PMMA plastic based micro-fluidic system made by precise machining. The activity of channel proteins incorporated into the planar membrane is successfully recorded.

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Surface Science Society Japan  

DOI： 10.1380/ejssnt.2005.79

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

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記述言語：英語   掲載種別：研究論文（国際会議プロシーディングス）   出版者・発行元：IEEE  

We report the novel methods to reconstitute artificial lipid membranes in an array on a chip, using the micro-fluidic system, for the batch analysis of membrane proteins. The micro-fabricated device should render the ease of injection/recirculation of the reagents as well as multi-channel and high-sensitive detection of protein activities incorporated in the bilayer membrane. It is shown that the lipid bilayer membrane can be formed on 100 similar to 200 mum square apertures fabricated in a silicon substrate in two methods. In the first method, the bilayer is formed by flowing the lipid solution and the buffer alternately. In the second method, it is done by placing a buffer droplet on a thin layer of the lipid solution. Parylene coating is found to be suitable for both bilayer formation and noise reduction. The future application includes the high-sensitive ion sensor chip and high-throughput drug screening chip.

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.44.S224_1

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ROYAL SOC CHEMISTRY  

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 mm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

DOI： 10.1039/b405967k

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

Except for the case of gradual activation of EF(1) (F-1-ATPase from Escherichia coli) caused by the dissociation of the epsilon subunit [Laget, P.P. and Smith, J.B. (1979) Arch. Biochem. Biophys. 197, 83-89], EF(1) has long been thought not to show a time-dependent change in activity [Senior, A.E. et al. (1992) Arch. Biochem. Biophys. 297, 340-344]. Here, we report the time-dependent inactivation and activation of EF(1), which are apparently similar to those of mitochondrial F-1-ATPases [Vasilyeva, E.A. et al, (1982) Biochem. J. 202, 15-23]. Analysis of these changes as a function of ATP concentrations in relation to negative cooperativity revealed that the initial inactivation phase was attributable to the decrease in the V-max associated with the low K-m (around 10 mu M), and the following activation, probably due to the dissociation of the epsilon subunit, corresponded to the increase in the V-max associated with the high K-m (in the order of 100 mu M). Thus, the time-dependent change in EF(1) activity is closely related to the apparent negative cooperativity (multiple K-m values) of ATP hydrolysis.

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