Hideo Akutsu

Osaka University, Suika, Ōsaka, Japan

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Publications (192)656.28 Total impact

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    ABSTRACT: FoF1-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the Foc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TFoc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TFoc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TFoc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H(+)-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D (13)C-(13)C correlation spectra of TFoc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C(α)i+1-C(α)i correlation spectrum of specifically (13)C,(15)N-labeled TFoc rings. The C(δ) chemical shift of Glu-56, which is essential for H(+) translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H(+)-locked conformation with Asn-23. The chemical shift of Asp-61 C(γ) of the E. coli c ring indicated an involvement of a water molecule in the H(+) locking, in contrast to the involvement of Asn-23 in the TFoc ring, suggesting two different means of proton storage in the c rings.
    Biophysical Journal 01/2014; 106(2):390-8. DOI:10.1016/j.bpj.2013.12.005 · 3.83 Impact Factor
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    ABSTRACT: ATP synthase produces ATP, a major energy source for metabolic processes in organisms, from ADP and inorganic phosphate in cellular membranes. ATP synthase is known as a rotary motor, in which the c-subunit ring functions as a rotor. In this work, we have tried to develop a more general preparation procedure of thermophilic -ring (-ring) for NMR measurements. The expression of is easily affected by various experimental conditions such as temperature, shape and size of a flask, a volume of medium, and shaking rate of an incubator. Accordingly, we have tried to optimize the expression conditions of . -rings were purified from according to a reported method. We modified purification procedures to improve purity and yield of . On top of them, we found a new combination of detergents for the purification at anion-exchange column chromatography. To examine the effect of lipid environments on the structure, the -rings were reconstituted into two kinds of lipid bilayers, namely, saturated and unsaturated lipid ones. Then, we have compared characteristics of the -ring structures in these membranes with solid-state NMR.
    12/2013; 17(2). DOI:10.6564/JKMRS.2013.17.2.067
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    ABSTRACT: Biomolecular NMR chemical shift data are key information for the functional analysis of biomolecules and the development of new techniques for NMR studies utilizing chemical shift statistical information. Structural genomics projects are major contributors to the accumulation of protein chemical shift information. The management of the large quantities of NMR data generated by each project in a local database and the transfer of the data to the public databases are still formidable tasks because of the complicated nature of NMR data. Here we report an automated and efficient system developed for the deposition and annotation of a large number of data sets including (1)H, (13)C and (15)N resonance assignments used for the structure determination of proteins. We have demonstrated the feasibility of our system by applying it to over 600 entries from the internal database generated by the RIKEN Structural Genomics/Proteomics Initiative (RSGI) to the public database, BioMagResBank (BMRB). We have assessed the quality of the deposited chemical shifts by comparing them with those predicted from the PDB coordinate entry for the corresponding protein. The same comparison for other matched BMRB/PDB entries deposited from 2001-2011 has been carried out and the results suggest that the RSGI entries greatly improved the quality of the BMRB database. Since the entries include chemical shifts acquired under strikingly similar experimental conditions, these NMR data can be expected to be a promising resource to improve current technologies as well as to develop new NMR methods for protein studies.
    Journal of Biomolecular NMR 06/2012; 53(4):311-20. DOI:10.1007/s10858-012-9641-6 · 3.31 Impact Factor
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    ABSTRACT: F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.
    Protein Expression and Purification 02/2012; 82(2):396-401. DOI:10.1016/j.pep.2012.02.005 · 1.51 Impact Factor
  • Biophysical Journal 01/2012; 102(3):711-. DOI:10.1016/j.bpj.2011.11.3860 · 3.83 Impact Factor
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    ABSTRACT: We report an approach to determining membrane peptides and membrane protein complex structures by magic-angle-spinning solid-state NMR and molecular dynamics simulation. First, an ensemble of low energy structures of mastoparan-X, a wasp venom peptide, in lipid bilayers was generated by replica exchange molecular dynamics (REMD) simulation with the implicit membrane/solvent model. Next, peptide structures compatible with experimental (13)C(α), C(β), and C' chemical shifts were selected from the ensemble. The (13)C(α) chemical shifts alone were sufficient for the selection with backbone rmsd's of ∼0.8 Å from the experimentally determined structure. The dipolar couplings between the peptide protons and lipid (2)H/(31)P nuclei were obtained from the (13)C-observed (2)H/(31)P-selective (1)H-demagnetization experiments for selecting the backbone and side chain structures relative to the membrane. The simulated structure agreed with the experimental one in the depth and orientation. The REMD simulation can be used for supplementing the limited structural constraints obtainable from the solid-state NMR spectra.
    The Journal of Physical Chemistry B 06/2011; 115(29):9327-36. DOI:10.1021/jp205290t · 3.38 Impact Factor
  • Bulletin of the Chemical Society of Japan 01/2011; 84(10):1096-1101. DOI:10.1246/bcsj.20110039 · 2.22 Impact Factor
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    ABSTRACT: The subunit c-ring of H(+)-ATP synthase (F(o) c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly [(13)C, (15)N]-labeled F(o) c from E. coli (EF(o) c) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the (13)C and (15)N signals were assigned. The obtained chemical shifts suggested that EF(o) c takes on a hairpin-type helix-loop-helix structure in membranes as in an organic solution. The results on the magnetization transfer between the EF(o) c and deuterated lipids indicated that Ile55, Ala62, Gly69 and F76 were lined up on the outer surface of the oligomer. This is in good agreement with the cross-linking results previously reported by Fillingame and his colleagues. This agreement reveals that the reconstituted EF(o) c oligomer takes on a ring structure similar to the intact one in vivo. On the other hand, analysis of the (13)C nuclei distance of [3-(13)C]Ala24 and [4-(13)C]Asp61 in the F(o) c-ring did not agree with the model structures proposed for the EF(o) c-decamer and dodecamer. Interestingly, the carboxyl group of the essential Asp61 in the membrane-embedded EF(o) c-ring turned out to be protonated as COOH even at neutral pH. The hydrophobic surface of the EF(o) c-ring carries relatively short side chains in its central region, which may allow soft and smooth interactions with the hydrocarbon chains of lipids in the liquid-crystalline state.
    Journal of Biomolecular NMR 09/2010; 48(1):1-11. DOI:10.1007/s10858-010-9432-x · 3.31 Impact Factor
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    ABSTRACT: Instrumentation for high-field dynamic nuclear polarization (DNP) at 14.1 T was developed to enhance the nuclear polarization for NMR of solids. The gyrotron generated 394.5 GHz submillimeter (sub-mm) wave with a power of 40 W in the second harmonic TE(0,6) mode. The sub-mm wave with a power of 0.5-3 W was transmitted to the sample in a low-temperature DNP-NMR probe with a smooth-wall circular waveguide system. The (1)H polarization enhancement factor of up to about 10 was observed for a (13)C-labeled compound with nitroxyl biradical TOTAPOL. The DNP enhancement was confirmed by the static magnetic field dependence of the NMR signal amplitude at 90 K. Improvements of the high-field DNP experiments are discussed.
    Physical Chemistry Chemical Physics 06/2010; 12(22):5799-803. DOI:10.1039/C002268C · 4.20 Impact Factor
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    ABSTRACT: Green sulfur photosynthetic bacteria optimize their antennas, chlorosomes, especially for harvesting weak light by organizing bacteriochlorophyll (BChl) assembly without any support of proteins. As it is difficult to crystallize the organelles, a high-resolution structure of the light-harvesting devices in the chlorosomes has not been clarified. We have determined the structure of BChl c assembly in the intact chlorosomes from Chlorobium limicola on the basis of (13)C dipolar spin-diffusion solid-state NMR analysis of uniformly (13)C-labeled chlorosomes. About 90 intermolecular C-C distances were obtained by the simultaneous assignment of distance correlations and the structure optimization preceded by the polarization-transfer matrix analysis. An atomic structure was obtained, using these distance constraints. The determined structure of the chlorosomal BChl c assembly is built with the parallel layers of piggyback-dimers. This supramolecular structure would provide insights into the mechanism of weak-light capturing.
    Photosynthesis Research 06/2010; 104(2-3):221-31. DOI:10.1007/s11120-009-9523-2 · 3.19 Impact Factor
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    ABSTRACT: The combined use of selective deuteration, stereo-array isotope labeling (SAIL), and fast magic-angle spinning effectively suppresses the 1H-1H dipolar couplings in organic solids. This method provided the high-field 1H NMR linewidths comparable to those achieved by combined rotation and multiple-pulse spectroscopy. This technique was applied to two-dimensional 1H-detected 1H-1H polarization transfer CHH experiments of valine. The signal sensitivity for the 1H-detected CHH experiments was greater than that for the 13C-detected 1H-1H polarization transfer experiments by a factor of 2-4. We obtained the 1H-1H distances in SAIL valine by CHH experiments with an accuracy of about 0.2A by using a theory developed for 1H-1H polarization transfer in 13C-labeled organic compounds.
    Journal of Magnetic Resonance 04/2010; 203(2):253-6. DOI:10.1016/j.jmr.2010.01.005 · 2.32 Impact Factor
  • Hideo Akutsu, Toshimichi Fujiwara
    Encyclopedia of Magnetic Resonance, 03/2010; , ISBN: 9780470034590
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    ABSTRACT: F(1)-ATPase, composed of alpha, beta, gamma, delta, and epsilon subunits, is a unique enzyme in terms of its rotational catalytic activity. The smallest unit showing this function is the alpha(3)beta(3)gamma complex. We have investigated the alpha(3)beta(3)gamma epsilon(Delta C) (epsilon(Delta C), truncated epsilon) complex from thermophilic Bacillus PS3 (TF(1)', 360 kDa) in the solution state by using the combination of extensive deuteration, segmental-labeling, and CRINEPT (cross-correlated relaxation-enhanced polarization transfer) NMR. Well-resolved CRINEPT-HMQC (heteronuclear multiple-quantum correlation) spectra of partially (15)N-labeled TF(1)' were obtained for this huge and asymmetric protein complex. The spectrum of the C-terminal domain of the beta subunit revealed that the open form of the beta subunit in the TF(1)' complex is similar to that of the free beta monomer. The open beta subunit in the TF(1)' complex does not exhibit high affinity for nucleotides unlike the monomer, but this is in agreement with the results of single-molecule analysis of TF(1)alpha(3)beta(3)gamma. On the other hand, the closed form of the beta subunit in the TF(1)' complex was shown to be distinct from that of the nucleotide-bound beta monomer. This is consistent with a previous report that the closed form of the TF(1)beta monomer could be a catalytically activated state. The loop between the N-terminal beta-barrel and the central domain is highly flexible in the TF(1)' complex, in contrast to that in the alpha(3)beta(3) hexamer, suggesting that it is affected by the presence of the gamma subunit in this area.
    Journal of Molecular Biology 03/2010; 398(2):189-99. DOI:10.1016/j.jmb.2010.03.013 · 3.96 Impact Factor
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    ABSTRACT: The epsilon subunit, a small subunit located in the F1 domain of ATP synthase and comprising two distinct domains, an N-terminal beta-sandwich structure and a C-terminal alpha-helical region, serves as an intrinsic inhibitor of ATP hydrolysis activity. This inhibitory function is especially important in photosynthetic organisms as the enzyme cannot synthesize ATP in the dark, but may catalyse futile ATP hydrolysis reactions. To understand the structure-function relationship of this subunit in F1 from photosynthetic organisms, we solved the NMR structure of the epsilon subunit of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, and examined the flexibility of the C-terminal domains using molecular dynamics simulations. In addition, we revealed the significance of the C-terminal alpha-helical region of the epsilon subunit in determining the binding affinity to the complex based on the assessment of the inhibition of ATPase activity by the cyanobacterial epsilon subunit and the chimaeric subunits composed of the N-terminal domain from the cyanobacterium and the C-terminal domain from spinach. The differences observed in the structural and biochemical properties of chloroplast and bacterial epsilon subunits explains the distinctive characteristics of the epsilon subunits in the ATPase complex of the photosynthetic organism.
    Biochemical Journal 09/2009; 425(1):85-94. DOI:10.1042/BJ20091247 · 4.78 Impact Factor
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    ABSTRACT: The rotation of F1-ATPase (F1) is driven by the open/close bending motion of the beta subunit. The mechanism underlying the bending motion was investigated for the F1beta monomer from thermophilic Bacillus PS3 (TF1beta) in solution, using mutagenesis and NMR. The hydrogen bond networks involving the side chains of Lys-164 (numbering for TF1beta; 162 for mitochondrial F1beta in parentheses), Thr-165(163), Arg-191(189), Asp-252(256), Asp-311(315), and Arg-333(337) in the catalytic region are significantly different for the ligand-bound and freebeta subunits in the crystal structures of mitochondrial F1. The role of each amino acid residue was examined by Ala substitution. beta(K164A) reduced the affinity constant for 5'-adenyl-beta,gamma-imidodiphosphate by 20-fold and abolished the conformational change associated with nucleotide binding and the ATPase activity of alpha3beta(K164A)3gamma.beta(T165A) and beta(D252A) exhibited no effect on the binding affinity but abolished the conformational change and the ATPase activity. The chemical shift perturbation of backbone amide signals of the segmentally labeled beta(mutant)s indicated stepwise propagation of the open/close conversion on ligand binding. The key action in the conversion is the switching of the hydrogen-bonding partner of Asp-252 from Lys-164 to Thr-165. Residual dipolar coupling analysis revealed that the closed conformation of the beta monomer was more closed than that in the crystal structure and was different for MgATP- and MgADP-bound beta subunits. Actually, MgATP induced a conformational change around Tyr-307 (311 for MF1beta), whereas MgADP did not. The significance of these findings is discussed in connection with the catalytic rotation of F1-ATPase.
    Journal of Biological Chemistry 12/2008; 284(4):2374-82. DOI:10.1074/jbc.M808212200 · 4.60 Impact Factor
  • Seikagaku. The Journal of Japanese Biochemical Society 11/2008; 80(10):907-16. · 0.04 Impact Factor
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    ABSTRACT: We have developed a theory for (1)H-(1)H distance measurements from the direct polarization transfer in (13)C-labeled solids under magic-angle spinning. The polarization transfer caused by the (1)H-(1)H dipolar interactions was analyzed with zeroth-order average Hamiltonian for a (1)H-(13)C-(13)C-(1)H spin system in the frame modulated by (13)C-(1)H dipolar interactions and chemical shifts. Strong (13)C-(1)H dipolar couplings primarily determine the recovery of the (1)H-(1)H coupling as a function of sample spinning frequency. The effect of additional (1)H spins on the polarization transfer was also taken into account. We have applied this method to the distance measurements for uniformly (13)C-, (15)N-labeled L-valine and adenosine. Experimental (1)H polarization transfer was monitored through high-resolution (13)C-NMR. The theoretical analysis provided the distances up to about 3 A with an accuracy of about 0.2 A and those of about 4 A with 1 A even from the transfer amplitudes at a few mixing times. The longer distances are partly affected by the relayed polarization transfer which makes apparent (1)H-(1)H distances shorter. Our theory based on the coherent polarization transfer in the initial build-up regime was compared to the description by the rate equations with spin diffusion time constants.
    The Journal of Chemical Physics 11/2008; 129(15):154504. DOI:10.1063/1.2993170 · 3.12 Impact Factor
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    ABSTRACT: Tetraheme cytochrome c 3 (cyt c 3) exhibits extremely low reduction potentials and unique properties. Since axial ligands should be the most important factors for this protein, every axial histidine of Desulfovibrio vulgaris Miyazaki F cyt c 3 was replaced with methionine, one by one. On mutation at the fifth ligand, the relevant heme could not be linked to the polypeptide, revealing the essential role of the fifth histidine in heme linking. The fifth histidine is the key residue in the structure formation and redox regulation of a c-type cytochrome. A crystal structure has been obtained for only H25M cyt c 3. The overall structure was not affected by the mutation except for the sixth methionine coordination at heme 3. NMR spectra revealed that each mutated methionine is coordinated to the sixth site of the relevant heme in the reduced state, while ligand conversion takes place at hemes 1 and 4 during oxidation at pH 7. The replacement of the sixth ligand with methionine caused an increase in the reduction potential of the mutated heme of 222-244 mV. The midpoint potential of a triheme H52M cyt c 3 is higher than that of the wild type by approximately 50 mV, suggesting a contribution of the tetraheme architecture to the lowering of the reduction potentials. The hydrogen bonding of Thr24 with an axial ligand induces a decrease in reduction potential of approximately 50 mV. In conclusion, the bis-histidine coordination is strategically essential for the structure formation and the extremely low reduction potential of cyt c 3.
    Biochemistry 10/2008; 47(36):9405-15. DOI:10.1021/bi8005708 · 3.19 Impact Factor
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    ABSTRACT: The F(1)F(o)-ATP synthase utilizes the transmembrane H(+) gradient for the synthesis of ATP. F(o) subunit c-ring plays a key role in transporting H(+) through F(o) in the membrane. We investigated the interactions of Escherichia coli subunit c with dimyristoylphosphatidylcholine (DMPC-d(54)) at lipid/protein ratios of 50:1 and 20:1 by means of (2)H-solid-state NMR. In the liquid-crystalline state of DMPC, the (2)H-NMR moment values and the order parameter (S(CD)) profile were little affected by the presence of subunit c, suggesting that the bilayer thickness in the liquid-crystalline state is matched to the transmembrane hydrophobic surface of subunit c. On the other hand, hydrophobic mismatch of subunit c with the lipid bilayer was observed in the gel state of DMPC. Moreover, the viscoelasticity represented by a square-law function of the (2)H-NMR relaxation was also little influenced by subunit c in the fluid phase, in contrast with flexible nonionic detergents or rigid additives. Thus, the hydrophobic matching of the lipid bilayer to subunit c involves at least two factors, the hydrophobic length and the fluid mechanical property. These findings may be important for the torque generation in the rotary catalytic mechanism of the F(1)F(o)-ATPse molecular motor.
    Biophysical Journal 07/2008; 94(11):4339-47. DOI:10.1529/biophysj.107.123745 · 3.83 Impact Factor

Publication Stats

3k Citations
656.28 Total Impact Points

Institutions

  • 1975–2014
    • Osaka University
      • • Institute for Protein Research
      • • Division of Protein Structural Biology
      Suika, Ōsaka, Japan
  • 2008
    • University of Wollongong
      City of Greater Wollongong, New South Wales, Australia
    • University of Wisconsin, Madison
      • Department of Biochemistry
      Madison, MS, United States
  • 2004–2007
    • Kwansei Gakuin University
      • School of Science and Technology
      Nishinomiya, Hyogo-ken, Japan
  • 2006
    • University of Hyogo
      Kōbe, Hyōgo, Japan
    • Kochi Medical School
      Kôti, Kōchi, Japan
  • 2002–2006
    • Nagaoka University of Technology
      • Department of Chemistry
      Нагаока, Niigata, Japan
  • 2005
    • RIKEN
      Вако, Saitama, Japan
  • 1987–2001
    • Yokohama National University
      Yokohama, Kanagawa, Japan
  • 2000
    • Tokyo Institute of Technology
      • Chemical Resources Laboratory
      Edo, Tōkyō, Japan
  • 1998
    • Mitsubishi Heavy Industries
      Edo, Tōkyō, Japan