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Toshiyuki Kohno

Publications and source records attributed to Toshiyuki Kohno.

32 records · Page 2Linked to original sources
PubMed2004

Structural insight into nucleotide recognition in tau-protein kinase I/glycogen synthase kinase 3 beta.

Human tau-protein kinase I (TPK I; also known as glycogen synthase kinase 3 beta; GSK3 beta) is a serine/threonine protein kinase that participates in Alzheimer's disease. Here, binary complex structures of full-length TPK I/GSK3 beta with the ATP analogues ADP and AMPPNP solved by the X-ray diffraction method at 2.1 and 1.8 A resolution, respectively, are reported. TPK I/GSK3 beta is composed of three domains: an N-terminal domain consisting of a closed beta-barrel structure, a C-terminal domain containing a 'kinase fold' structure and a small extra-domain subsequent to the C-terminal domain. The catalytic site is between the two major domains and has an ATP-analogue molecule in its ATP-binding site. The adenine ring is buried in the hydrophobic pocket and interacts specifically with the main-chain atoms of the hinge loop. The overall structure and substrate-binding residues are similar to those observed in other Ser/Thr protein kinases, while Arg141 (which is not conserved among other Ser/Thr protein kinases) is one of the key residues for specific ATP/ADP recognition by TPK I/GSK3 beta. No residues are phosphorylated, while the orientation of the activation loop in TPK I/GSK3 beta is similar to that in phosphorylated CDK2 and ERK2, suggesting that TPK I/GSK3 beta falls into a conformation that enables it to be constitutively active.

Adenosine Diphosphate↗
PubMed2004

Solution structure and functional characterization of SGTx1, a modifier of Kv2.1 channel gating.

SGTx1 is a peptide toxin isolated from the venom of the spider Scodra griseipes that has been shown to inhibit outward K(+) currents in rat cerebellar granule neurons. Although its amino acid sequence is known to be highly (76%) homologous with that of hanatoxin (HaTx), a well-characterized modifier of Kv2.1 channel gating, the structural and functional characteristics of SGTx1 remain largely unknown. Here we describe the NMR solution structure of SGTx1, the mechanism of its interaction with Kv2.1 channels, and its effect on channel activity once bound. The NMR structure of SGTx1 contains a molecular fold closely resembling the "inhibitor cystine knot" of HaTx, which is composed of an antiparallel beta-sheet and four chain reversals stabilized by three disulfide bonds. Functionally, SGTx1 reversibly inhibited K(+) currents in oocytes expressing Kv2.1 channels. Moreover, generation of steady-state activation curves showed that, consistent with other gating modifiers, SGTx1 acted by shifting the activation of the channel to more depolarized voltages. Thus, the surface profile and mechanism of action of SGTx1 are similar to those of HaTx. Still, detailed comparison of SGTx1 with HaTx revealed differences in binding affinity and conformational homogeneity that result from differences in the charge distribution at the binding surface and in the amino acid composition of the respective beta-hairpin structures in the peptides.

Amino Acid Sequence↗
PubMed2004

Signal assignments and chemical-shift structural analysis of uniformly 13C, 15N-labeled peptide, mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning.

Carbon-13 and nitrogen-15 signals of fully isotope-labeled 15-residue peptide, glycinated mastoparan-X, in a solid state were assigned by two- and three-dimensional NMR experiments under magic-angle spinning conditions. Intra-residue spin connectivities were obtained with multidimensional correlation experiments for C'-C(alpha)-C(beta) and N-C(alpha)-C(beta). Sequence specific assignments were performed with inter-residue C(alpha)-C(alpha) and N-C(alpha)C(beta) correlation experiments. Pulse sequences for these experiments have mixing periods under recoupled zero- and double-quantum (13)C-(13)C and (15)N-(13)C dipolar interactions. These correlation spectra allowed the complete assignments of (13)C and (15)N backbone and (13)C(beta) signals. Chemical shift analysis of the (13)C and (15)N signals based on empirical and quantum chemical databases for proteins indicated that the backbone between residues 3 and 14 forms alpha-helix and residue 2 has extended conformation in the solid state. This structure was compared with the G-protein- and membrane-bound structures of mastoparan-X.

Carbon Isotopes↗
PubMed2004

A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones. In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.

Amino Acid Sequence↗
PubMed2003

Channel-forming membrane permeabilization by an antibacterial protein, sapecin: determination of membrane-buried and oligomerization surfaces by NMR.

The action mechanism of sapecin, an antibacterial peptide with membrane permeabilization activity, was investigated. The dose dependence of the membrane permeabilization caused by sapecin was sigmoidal, suggesting that sapecin oligomerization leads to the membrane permeabilization. Solution nuclear magnetic resonance analysis of the sapecin-phospholipid vesicle complex revealed the surface buried in the membrane and oligomerization surface on the sapecin molecule. The membrane-buried surface of sapecin was determined by observing the transferred cross-saturation phenomena from the alkyl chains of the phospholipid vesicle to the amide protons of sapecin. The membrane-buried surface contains basic and highly exposed hydrophobic residues, which are suitable for interacting with the acidic bacterial membrane. The oligomerization surface was also identified by comparisons between the results from hydrogen-deuterium exchange experiments and transferred cross-saturation experiments. On the basis of the results from the NMR experiments we built a putative model of sapecin oligomers, which provides insights into the membrane permeabilization caused by insect defensins.

Amino Acid Sequence↗
PubMed2003

Influence of fragment reaction of relativistic heavy charged particles on heavy-ion radiotherapy.

The production of projectile fragments is one of the most important, but not yet perfectly understood, problems to be considered when planning for the utilization of high-energy heavy charged particles for radiotherapy. This paper reports our investigation of the fragments' fluence and linear energy transfer (LET) spectra produced from various incident ions using an experimental approach to reveal these physical qualities of the beams. Polymethyl methacrylate, as a substitute for the human body, was used as a target. A deltaE-E counter telescope with a plastic scintillator and a BGO scintillator made it possible to identify the species of fragments based on differences of various elements. By combining a gas-flow proportional counter with a counter telescope system, LET spectra as well as fluence spectra of the fragments were derived for each element down from the primary particles to hydrogen. Among them, the information on hydrogen and helium fragments was derived for the first time. The result revealed that the number of light fragments, such as hydrogen and helium, became larger than the number of primaries in the vicinity of the range end. However, the greater part of the dose delivered to a cell was still governed by the primaries. The calculated result of a simulation used for heavy-ion radiotherapy indicated room for improving the reaction model.

Ions↗
PubMed2003

Electron density measurement with dual-energy x-ray CT using synchrotron radiation.

Monochromatic x-ray computed tomography (CT) at two different energies provides information about electron density of human tissue without ambiguity due to the beam hardening effect. This information makes the treatment planning for proton and heavy-ion radiotherapy more precise. We have started a feasibility study on dual energy x-ray CT by using synchrotron radiation. A translation-rotation scanning CT system was developed for quantitative measurement in order to clarify what precision in the measurement was achieved. Liquid samples of solutions of K2HPO4 and solid samples of tissue equivalent materials were used to simulate human tissue. The experiments were carried out using monochromatic x-rays with energies of 40, 70 and 80 keV produced by monochromatizing synchrotron radiation. The solid samples were also measured in a complementary method using high-energy carbon beams to evaluate the electron densities. The measured electron densities were compared with the theoretical values or the values measured in the complementary method. It was found that these values were in agreement in 0.9% on average. Effective atomic numbers were obtained as well from dual-energy x-ray CT. The tomographic image based on each of the electron densities and the effective atomic number presents a different feature of the material, and its contrast drastically differs from that in a conventional CT image.

Connective Tissue↗
PubMed2003

Structural basis of the KcsA K(+) channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method.

We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K(+) channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K(+) channel. Based on properties of the AgTx2 binding site with the KcsA K(+) channel, we present a surface motif that is observed in pore-blocking toxins affecting the K(+) channel. Furthermore, we also explain the structural basis of the specificity of the K(+) channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.

Amino Acid Motifs↗
PubMed2003

A wheat germ cell-free system is a novel way to screen protein folding and function.

For high-throughput protein structural analysis, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as that involving Escherichia coli cells, have been developed, the number of overexpressed proteins showing the same biological activities as those of the native proteins is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the proteins functioning in solution were synthesized as soluble forms. This suggests the applicability of this protein synthesis method to determination of the solution structures of functional proteins. To examine this possibility, we have synthesized two (15)N-labeled proteins and obtained (1)H-(15)N HSQC spectra for them. The structural analysis of these proteins has already progressed with an E. coli overexpression system, and (1)H-(15)N HSQC spectra for biologically active proteins have already been obtained. Comparing the spectra, we have shown that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity. This is the first experimental evidence of the applicability of the wheat germ cell-free protein synthesis system to high-throughput protein structural analysis.

Cell-Free System↗
PubMed2002

Three-dimensional solution structure of the sodium channel agonist/antagonist delta-conotoxin TxVIA.

The three-dimensional solution structure of delta-conotoxin TxVIA, a 27-mer peptide agonist/antagonist of sodium channels, was determined by two-dimensional (1)H NMR spectroscopy with simulated annealing calculations. A total of 20 converged structures of delta-conotoxin TxVIA were obtained on the basis of 360 distance constraints obtained from nuclear Overhauser effect connectivities, 28 torsion angle constraints, and 27 constraints associated with hydrogen bonds and disulfide bonds. The atomic root mean square difference about the averaged coordinate positions is 0.35 +/- 0.07 A for the backbone atoms (N, C(alpha), C) and 0.98 +/- 0.14 A for all heavy atoms of the entire peptide. The molecular structure of delta-conotoxin TxVIA is composed of a short triple-stranded antiparallel beta-sheet. The overall beta-sheet topology is +2x, -1, which is the same as those for other conotoxins. However, the three-dimensional structure of delta-conotoxin TxVIA has an unusual hydrophobic patch on one side of the molecule, which may play an important role in the sodium channel binding. These results provide a molecular basis for understanding the mechanism of sodium channel modulation through the toxin-channel interaction and insight into the discrimination of different ion channels.

Amino Acid Sequence↗
PubMed2002

Charge conversion enables quantification of the proximity between a normally-neutral mu-conotoxin (GIIIA) site and the Na+ channel pore.

mu-Conotoxin (mu-CTX) inhibits Na+ flux by obstructing the Na+ channel pore. Previous studies of mu-CTX have focused only on charged toxin residues, ignoring the neutral sites. Here we investigated the proximity between the C-terminal neutral alanine (A22) of mu-CTX and the Na+ channel pore by replacing it with the negatively charged glutamate. The analog A22E and wild-type (WT) mu-CTX exhibited identical nuclear magnetic resonance spectra except at the site of replacement, verifying that they have identical backbone structures. A22E significantly reduced mu-CTX affinity for WT mu1 Na+ channels (90-fold), as if the inserted glutamate repels the anionic pore receptor. We then looked for the interacting partner(s) of residue 22 by determining the potency of block of Y401K, Y401A, E758Q, D762K, D762A, E765K, E765A and D1241K channels by WT mu-CTX and A22E, followed by mutant cycle analysis to assess their individual couplings. Our results show that A22E interacts strongly with E765K from domain II (DII) (deltadeltaG=2.2 +/- 0.1 vs. <1 kcal/mol for others). We conclude that mu-CTX residue 22 closely associates with the DII pore in the toxin-bound channel complex. The approach taken may be further exploited to study the proximity of other neutral toxin residues with the Na+ channel pore.

Alanine↗
PubMed2000

[Fundamental Study on Heavy Ion CT Using Pencil Beam Scanning Method]

The feasibility of heavy ion computed tomography was investigated. A cylindrical polyethylene sample of 50 mm in diameter was used to establish the pencil beam scanning method and estimate the performance of the system. The sample was irradiated with carbon ions of 290 MeV/u and 400 MeV/u, and helium ions of 150 MeV/u. The residual beam energy was measured by a BGO scintillator in coincidence with two small plastic scintillators placed in front of and behind the sample to restrict the beam path and improve the spatial resolution in the CT reconstruction. The projection data were obtained by moving the sample only in the transverse direction because of its symmetrical structure. A Lucite sample with many holes of different diameters was used to demonstrate the spatial resolution of this CT system. From the reconstructed images the spatial resolution was estimated to be less than 2 mm under the conditions in this work. The electron density ratios of ethyl alcohol, water, and Lucite to polyethylene were obtained, which are in good agreement with the calculated values.

Journal Article↗