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Hideo Akutsu

Publications and source records attributed to Hideo Akutsu.

At least 19 recordsLinked to original sources
PubMed2007

Structure of the light-harvesting bacteriochlorophyll c assembly in chlorosomes from Chlorobium limicola determined by solid-state NMR.

We have determined the atomic structure of the bacteriochlorophyll c (BChl c) assembly in a huge light-harvesting organelle, the chlorosome of green photosynthetic bacteria, by solid-state NMR. Previous electron microscopic and spectroscopic studies indicated that chlorosomes have a cylindrical architecture with a diameter of approximately 10 nm consisting of layered BChl molecules. Assembly structures in huge noncrystalline chlorosomes have been proposed based mainly on structure-dependent chemical shifts and a few distances acquired by solid-state NMR, but those studies did not provide a definite structure. Our approach is based on (13)C dipolar spin-diffusion solid-state NMR of uniformly (13)C-labeled chlorosomes under magic-angle spinning. Approximately 90 intermolecular C C distances were obtained by simultaneous assignment of distance correlations and structure optimization preceded by polarization-transfer matrix analysis. It was determined from the approximately 90 intermolecular distances that BChl c molecules form piggyback-dimer-based parallel layers. This finding rules out the well known monomer-based structures. A molecular model of the cylinder in the chlorosome was built by using this structure. It provided insights into the mechanisms of efficient light harvesting and excitation transfer to the reaction centers. This work constitutes an important advance in the structure determination of huge intact systems that cannot be crystallized.

Bacterial Proteins↗
PubMed2006

Process of maturation of tetraheme cytochrome c3 in a Shewanella expression system.

The process of maturation of multiheme proteins is not yet well known, while that of monoheme ones has been relatively well investigated. Two kinds of partly unfolded tetraheme cytochrome c3 were obtained on overexpression in Shewanella oneidensis TSP-C. These proteins were characterized by circular dichroism and nuclear magnetic resonance spectroscopy. It turned out that the tetraheme architecture, and the fifth and sixth ligand coordination are almost mature, while some parts of the polypeptide are unfolded. The unfolded residues are mainly located in the helix-rich region including heme attachment and axial ligand sites. This suggests that the formation of the heme architecture, coordination of axial ligands and helix formation should be coupled with each other. While the former two can take place automatically, the helix formation would need help by a chaperone-like function in the cytochrome c maturation (Ccm) machinery. It must be working in sulphate-reducing bacteria. The Ccm machinery in S. oneidensis is likely insufficient to help the maturation of proteins with cyclic heme architectures. This is the first report providing an insight into the process of maturation of tetraheme cytochrome c3.

Circular Dichroism↗
PubMed2006

3D structure of amyloid protofilaments of beta2-microglobulin fragment probed by solid-state NMR.

Understanding the structure and formation of amyloid fibrils, the filamentous aggregates of proteins and peptides, is crucial in preventing diseases caused by their deposition and, moreover, for obtaining further insight into the mechanism of protein folding and misfolding. We have combined solid-state NMR, x-ray fiber diffraction, and atomic force microscopy to reveal the 3D structure of amyloid protofilament-like fibrils formed by a 22-residue K3 peptide (Ser(20)-Lys(41)) of beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis. Although a uniformly (13)C,(15)N-labeled sample was used for the NMR measurements, we could obtain the 3D structure of the fibrils on the basis of a large number of structural constraints. The conformation of K3 fibrils was found to be a beta-strand-loop-beta-strand with each K3 molecule stacked in a parallel and staggered manner. It is suggested that the fibrillar conformation is stabilized by intermolecular interactions, rather than by intramolecular hydrophobic packing as seen in globular proteins. Together with thermodynamic studies of the full-length protein, formation of the fibrils is likely to require side chains on the intermolecular surface to pack tightly against those of adjacent monomers. By revealing the structure of beta(2)-microglobulin protofilament-like fibrils, this work represents technical progress in analyzing amyloid fibrils in general through solid-state NMR.

Amyloid↗
PubMed2006

Signal assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H +-ATP synthase subunit c, by magic-angle spinning solid-state NMR.

Signal assignment and secondary structural analysis of uniformly [13C, 15N] labeled H+-ATP synthase subunit c from E. coli (79 residues) in the solid state were carried out by two- and three-dimensional solid-state NMR under magic-angle spinning. The protein took on a unique structure even in the solid state from the 13C linewidths of about 1.7 ppm. On the basis of several inter- and intra-residue 13C-13C and 13C-15N chemical shift correlations, 78% of Calpha, 72% of Cbeta, 62% of C' and 61% of NH signals were assigned, which provided the secondary structure information for 84% of the 79 residues. Here, inter-residue correlations involving Gly, Ala, Pro and side-chains and a higher resolution in the 3D spectrum were significantly useful for the sequence specific assignment. On top of this, the 13C-13C correlation spectra of subunit c was analyzed by reproducing experimental cross peaks quantitatively with chemical shift prediction and signal-intensity calculation based on the structure. It revealed that the subunit c in the solid state could be specified by alpha-helices with a loop structure in the middle (at sequence 41-45) as in the case of the solution structure in spite of additional extended conformations at 76-79 at the C-terminus.

Amino Acid Sequence↗
PubMed2006

Detection of peptide-phospholipid interaction sites in bilayer membranes by 13C NMR spectroscopy: observation of 2H/31P-selective 1H-depolarization under magic-angle spinning.

We have developed a solid-state NMR method for observing the signals due to 13C spins of a peptide in the close vicinity of 31P and 2H spins in deuterated phospholipid bilayers. The signal intensities in 13C high-resolution NMR spectra directly indicate the depolarization of 1H by 1H-31P and 1H-2H dipolar couplings under multiple-contact cross-polarization. This method was applied to a fully 13C-, 15N-labeled 14-residue peptide, mastoparan-X (MP-X), bound to phospholipid bilayers whose fatty acyl chains are deuterated. The 13C NMR spectra for the depolarization were simulated from the chemical shifts and structure of membrane-bound MP-X previously determined and the distribution of 2H and 31P spins in lipid bilayers. The minimization of RMSD between the simulated and the experimental spectra showed that the amphiphilic alpha-helix of MP-X was located in the interface between the water layer and the hydrophobic domain of the bilayer, with nonpolar residues facing the phosphorus atoms and alkyl chains of the lipids.

Carbon Isotopes↗
PubMed2006

Structure of the cadherin-related neuronal receptor/protocadherin-alpha first extracellular cadherin domain reveals diversity across cadherin families.

The recent explosion in genome sequencing has revealed the great diversity of the cadherin superfamily. Within the superfamily, protocadherins, which are expressed mainly in the nervous system, constitute the largest subgroup. Nevertheless, the structures of only the classical cadherins are known. Thus, to broaden our understanding of the adhesion repertoire of the cadherin superfamily, we determined the structure of the N-terminal first extracellular cadherin domain of the cadherin-related neuronal receptor/protocadherin-alpha4. The hydrophobic pocket essential for homophilic adhesiveness in the classical cadherins was not found, and the functional significance of this structural domain was supported by exchanging the first extracellular cadherin domains of protocadherin and classical cadherin. Moreover, potentially crucial variations were observed mainly in the loop regions. These included the protocadherin-specific disulfide-bonded Cys-X(5)-Cys motif, which showed Ca(2+)-induced chemical shifts, and the RGD motif, which has been suggested to be involved in heterophilic cell adhesion via the active form of beta1 integrin. Our findings reveal that the adhesion repertoire of the cadherin superfamily is far more divergent than would be predicted by studying the classical cadherins alone.

Amino Acid Sequence↗
PubMed2006

Assembly of a mixture of isomeric BChl c from Chlorobium limicola as determined by intermolecular 13C-13C dipolar correlations: coexistence of dimer-based and pseudo-monomer-based stackings.

A mixture of bacteriochlorophyll (BChl) c isomers was extracted from the cells of Chlorobium limicola that were grown in the media of 13C-enriched and natural-abundance isotopic compositions. The magic-angle spinning 13C NMR proton-driven spin-diffusion spectra were recorded with mixing times of 50, 100, and 250 ms for two different kinds of in vitro aggregates, one consisting of pure [13C]BChl c and the other consisting of a 1:1 mixture of [13C]BChl c and [12C]BChl c; those peaks whose intensities were reduced to approximately 1/4 by this dilution were assigned to intermolecular 13C-13C dipolar correlation peaks. On the other hand, the nearest-neighbor intermolecular carbon-carbon close contacts with distances of 4-6 A were simulated, to predict observed correlation peaks, for six different models of BChl c assembly. They include weakly overlapped monomers forming structure 1 and structure 2, strongly overlapped dimers forming straight and inclined columns, and weakly overlapped dimers forming aligned and displaced layers. Comparison between the observed correlation peaks and the predicted carbon-carbon close contacts, for both the macrocycles and the side chains, led us to a conclusion that the weakly overlapped dimers forming displaced layers are most likely the assembly of the BChl c molecules in the aggregate.

Bacterial Proteins↗
PubMed2006

Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR.

The structure of mastoparan-X (MP-X), a G-protein activating peptide from wasp venom, in the state tightly bound to anionic phospholipid bilayers was determined by solid-state NMR spectroscopy. Carbon-13 and nitrogen-15 NMR signals of uniformly labeled MP-X were completely assigned by multidimensional intraresidue C-C, N-CalphaCbeta, and N-Calpha-C', and interresidue Calpha-CalphaCbeta, N-CalphaCbeta, and N-C'-Calpha correlation experiments. The backbone torsion angles were predicted from the chemical shifts of 13C', 13Calpha, 13Cbeta, and 15N signals with the aid of protein NMR database programs. In addition, two 13C-13C and three 13C-15N distances between backbone nuclei were precisely measured by rotational resonance and REDOR experiments, respectively. The backbone structure of MP-X was determined from the 26 dihedral angle restraints and five distances with an average root-mean-square deviation of 0.6 A. Peptide MP-X in the bilayer-bound state formed an amphiphilic alpha-helix for residues Trp3-Leu14 and adopted an extended conformation for Asn2. This membrane-bound conformation is discussed in relation to the peptide's activities to form pores in membranes and to activate G-proteins. This study demonstrates the power of multidimensional solid-state NMR of uniformly isotope-labeled molecules and distance measurements for determining the structures of peptides bound to lipid membranes.

GTP-Binding Proteins↗
PubMed2006

Effects of thermal treatments on the recovery of adsorbed water and photocatalytic activities of TiO2 photocatalytic systems.

The effects of thermal treatments on the rehydration process and photocatalytic activity were investigated by 1H NMR spectroscopy for six anatase abundant TiO2 photocatalysts with different properties. Acetic acid and benzoic acid were employed for photodecomposition in aqueous suspension. After the calcinations at 973 K, physisorbed water layers recovered relatively fast for P25, F4, and AMT-600 (shorter than 24 h) with no significant enhancement of the photocatalytic decomposition. On the other hand, for ST-01, UV-100, and AMT-100, the recovery was very slow (longer than 1 week) and only partially reversible, and the photocatalytic decomposition was considerably enhanced but retarded with rehydration. In the presence of adsorbed water, the binding of a carboxyl group of the molecules with adsorbed water is considered to compete with the direct adsorption on the surface, which reduces the amount of the direct adsorption and results in the reduction in the photocatalytic efficiency. In addition, the photocatalytic decomposition of benzoic acid with an aromatic ring was much faster in all of the TiO2 aqueous suspensions and more enhanced for the fully dehydroxylated TiO2 than that of acetic acid. These results suggest that the most efficient photocatalytic sites should be the hydrophobic sites on the TiO2 surface. The difference among the rehydration rates of different TiO2 is discussed in terms of thermally induced changes of surface morphology.

Absorption↗
PubMed2006

Specific binding of CO to tetraheme cytochrome c3.

Carbon monoxide (CO) has been identified as another bioactive molecule like NO. Binding of CO to a tetraheme cytochrome c(3) (cyt c(3)) was investigated using visible absorption spectroscopy, circular dichroism (CD), and NMR. CO was found to bind to the four hemes in different manners. CD spectra, however, indicated that only single-site CO binding can keep the protein intact. The K(d) for the single-site binding was 8.0 microM, which is a typical value for a CO sensor protein. Furthermore, NMR spectra of uniformly (15)N-labeled and specifically [(15)N]His-labeled proteins have provided evidence that CO specifically binds to the sixth coordination site of heme 2 via single-site binding. The CO-bound cyt c(3) could conduct redox reactions. In light of triheme cytochrome c(7), the CO-bound cyt c(3) may work as an electron transporter. It was reported for sulfate-reducing bacteria that CO can be used as an energy source and CO cycling is operating like H(2) cycling. Therefore, the CO-bound cyt c(3) may play a role in maintaining electron transport pathways on accumulation of toxic CO for its utilization.

Binding Sites↗
PubMed2006

Redox interaction of cytochrome c3 with [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F.

Cytochrome c3 isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between cytochrome c3 and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly 15N-labeled ferric cytochrome c3 except N-terminus, Pro, and Gly73 were assigned. 1H-15N HSQC spectra were recorded for 15N-labeled ferric and ferrous cytochrome c3, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes 1 and 3 in ferrous cytochrome c3 was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous cytochrome c3. To investigate the transient complex of cytochrome c3 and hydrogenase in detail, modeling of the complex was performed for the oxidized proteins using a docking program, ZDOCK 2.3, and NMR data. Furthermore, the roles of lysine residues of cytochrome c3 in the interaction with hydrogenase were investigated by site-directed mutagenesis. When the lysine residues around heme 4 were replaced by an uncharged residue, methionine, one by one, the Km of the electron-transfer kinetics increased. The results showed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 are important for formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome c3 reduction. This finding is consistent with the most possible structure of the transient complex obtained by modeling.

Binding Sites↗
PubMed2006

NMR study of the electron transfer complex of plant ferredoxin and sulfite reductase: mapping the interaction sites of ferredoxin.

Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.

Aspartic Acid↗
PubMed2006

A new solution structure of ATP synthase subunit c from thermophilic Bacillus PS3, suggesting a local conformational change for H+-translocation.

In F(o)F(1)-ATP synthase, an oligomer ring of F(o)c subunits acts as a rotary proton channel of the F(o)-proton motor. On the basis of the solution structure of the Escherichia coli F(o)c (EF(o)c) monomer, the rotation of the C-terminal helix coupled with the reorientation of the essential Asp61 side-chain on deprotonation was proposed to drive rotation of the whole c-ring. We have determined the NMR structure of F(o)c from thermophilic Bacillus PS3, TF(o)c, in an organic solvent mixture (chloroform/methanol (3:1, v/v)). Our results showed that, independent of pH, the carboxyl group of the essential Glu56 of TF(o)c protrudes toward the outside of the hairpin, a third orientation that differs from either of the two orientations in EF(o)c. Therefore, it would be inappropriate to draw conclusions about the mechanism of c-ring rotation on the basis of the conformations observed only for EF(o)c. The appearance of different hairpin structures shows that there are multiple energy minima for the hairpin structure in terms of helix rotation and axial displacement. The multiple energy minima may also provide a base for the different oligomeric states in the c-ring structure. A rotation mechanism of the F(o) motor coupled with H(+)-translocation is discussed on the basis of these results and the recently reported crystal structure of the c-ring from Ilyobacter tartaricus Na(+)-ATPase.

Amino Acid Sequence↗
PubMed2006

Orexin-A is composed of a highly conserved C-terminal and a specific, hydrophilic N-terminal region, revealing the structural basis of specific recognition by the orexin-1 receptor.

Orexins-A and B, also called hypocretins-1 and 2, respectively, are neuropeptides that regulate feeding and sleep-wakefulness by binding to two orphan G protein-coupled receptors named orexin-1 (OX(1)R) and orexin-2 (OX(2)R). The sequences and functions of orexins-A and B are similar to each other, but the high sequence homology (68%) is limited in their C-terminal half regions (residues 15-33). The sequence of the N-terminal half region of orexin-A (residues 1-14), containing two disulfide bonds, is very different from that of orexin-B. The structure of orexin-A was determined using two-dimensional homonuclear and (15)N and (13)C natural abundance heteronuclear NMR experiments. Orexin-A had a compact conformation in the N-terminal half region, which contained a short helix (III:Cys6-Gln9) and was fixed by the two disulfide bonds, and a helix-turn-helix conformation (I:Leu16-Ala23 and II:Asn25-Thr32) in the remaining C-terminal half region. The C-terminal half region had both hydrophobic and hydrophilic residues, which existed on separate surfaces to provide an amphipathic character in helices I and II. The nine residues on the hydrophobic surface are also well conserved in orexin-B, and it was reported that the substitution of each of them with alanine resulted in a significant drop in the functional potency at the receptors. Therefore, we suggest that they form the surface responsible for the main hydrophobic interaction with the receptors. On the other hand, the residues on the hydrophilic surface, together with the hydrophilic residues in the N-terminal half region that form a cluster, are known to make only small contributions to the binding to the receptors through similar alanine-scan experiments. However, since our structure of orexin-A showed that large conformational and electrostatical differences between orexins-A and B were rather concentrated in the N-terminal half regions, we suggest that the region of orexin-A is important for the preference for orexin-A of OX(1)R.

Amino Acid Sequence↗
PubMed2006

Identification of the substrate interaction region of the chitin-binding domain of Streptomyces griseus chitinase C.

Chitinase C from Streptomyces griseus HUT6037 was discovered as the first bacterial chitinase in family 19 other than chitinases found in higher plants. Chitinase C comprises two domains: a chitin-binding domain (ChBD(ChiC)) for attachment to chitin and a chitin-catalytic domain for digesting chitin. The structure of ChBD(ChiC) was determined by means of 13C-, 15N-, and 1H-resonance nuclear magnetic resonance (NMR) spectroscopy. The conformation of its backbone comprised two beta-sheets composed of two and three antiparallel beta-strands, respectively, this being very similar to the backbone conformations of the cellulose-binding domain of endoglucanase Z from Erwinia chrysanthemi (CBD(EGZ)) and the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12 (ChBD(ChiA1)). The interaction between ChBD(ChiC) and hexa-N-acetyl-chitohexaose was monitored through chemical shift perturbations, which showed that ChBD(ChiC) interacted with the substrate through two aromatic rings exposed to the solvent as CBD(EGZ) interacts with cellulose through three characteristic aromatic rings. Comparison of the conformations of ChBD(ChiA1), ChBD(ChiC), and other typical chitin- and cellulose-binding domains, which have three solvent-exposed aromatic residues responsible for binding to polysaccharides, has suggested that they have adopted versatile binding site conformations depending on the substrates, with almost the same backbone conformations being retained.

Amino Acid Sequence↗
PubMed2005

Phospholipid-dependent regulation of cytochrome c3-mediated electron transport across membranes.

Cytochrome c3 (cyt c3) can mediate electron transport across phosphatidylcholine (PC)/cardiolipin (CL) and PC/phosphatidylglycerol (PG) membranes. A two-molecule process is involved in the electron transport across PC/CL membranes in the liquid-crystalline state. In contrast, a single-molecule process dominates the electron transport across PC/CL membranes in the gel state and PC/PG membranes in the liquid-crystalline and gel states. Namely, the electron transport mechanism differs with the phospholipid composition and membrane fluidity. The rate-limiting step of the two-molecule process was lateral diffusion of cyt c3 in membranes. The rate constants for the three single-molecule process cases were similar to each other. To elucidate these reaction processes, interactions between cyt c3 and phosphate groups and between cyt c3 and the glycerol backbones of phospholipid bilayers were investigated by means of 31P and 2H solid-state NMR, respectively, for CL and PC/CL membranes. The results showed that the polar headgroups of both phosphatidylcholine and CL are involved in the binding of cyt c3. Also, cyt c3 penetrates into membranes, which would induce distortion of the lipid bilayer. The molecular mechanisms underlying the single- and two-molecule processes are discussed in terms of membrane structure.

Biological Transport↗