Shun Hirota

Publications of Shun Hirota

• Creation of an artificial metalloprotein with a Hoveyda-Grubbs catalyst moiety through the intrinsic inhibition mechanism of α-chymotrypsin.

  Authors: Takashi Matsuo, Chie Imai, Takefumi Yoshida, Takashi Saito, Takashi Hayashi, Shun Hirota

  Chemical communications (Cambridge, England). 01/2012; 48(11):1662-4.

  An L-phenylalanyl chloromethylketone-based inhibitor equipped with a Hoveyda-Grubbs catalyst moiety was regioselectively incorporated into the cleft of α-chymotrypsin through the intrinsic inhibitionAn L-phenylalanyl chloromethylketone-based inhibitor equipped with a Hoveyda-Grubbs catalyst moiety was regioselectively incorporated into the cleft of α-chymotrypsin through the intrinsic inhibition mechanism of the protein to construct an artificial organometallic protein.

• DNA cleavage by the photocontrolled cooperation of Zn(II) centers in an azobenzene-linked dizinc complex.

  Authors: Anangamohan Panja, Takashi Matsuo, Satoshi Nagao, Shun Hirota

  Inorganic chemistry. 11/2011; 50(22):11437-45.

  We synthesized a new photoactive dinuclear zinc(II) complex by linking two zinc centers with a ligand containing an azobenzene chromophore and investigated the DNA cleavage activities of its transWe synthesized a new photoactive dinuclear zinc(II) complex by linking two zinc centers with a ligand containing an azobenzene chromophore and investigated the DNA cleavage activities of its trans and cis forms. The trans structure of the dinuclear zinc complex was determined by X-ray crystallography, where each zinc center is situated in an octahedral coordination environment comprised of three nitrogen atoms from the ligand and three oxygen atoms from two nitrate ions. The dinuclear zinc complex containing the azobenzene chromophore was photoisomerizable between the trans and cis forms. The binding affinities of the trans and cis complexes with calf thymus (CT)-DNA were similar. Although the DNA cleavage activity of the trans complex was negligible, the cis complex was able to cleave DNA. We attribute the efficient activity of the cis complex to the cooperation of the two closely located zinc centers and the inactivity of the trans complex to the two metal centers positioned far away from each other. The DNA cleavage activity of the cis complex exhibited a pH-dependent bell-shaped profile, which has been observed in the hydrolytic cleavage of DNA by zinc complexes. The DNA cleavage activity was not inhibited by a major groove binder, methyl green, but decreased significantly by a minor groove binder, 4',6-diamidino-2-phenylindole, indicating that the dinuclear zinc complex binds to the minor groove of DNA. The present work shows the importance of the cooperation of two zinc ions for hydrolytic DNA cleavage, which can be photoregulated by linking the two metal centers with a photoisomerizable spacer, such as an azobenzene chromophore.

• Peroxidase activity enhancement of horse cytochrome c by dimerization.

  Authors: Zhonghua Wang, Takashi Matsuo, Satoshi Nagao, Shun Hirota

  Organic & biomolecular chemistry. 07/2011; 9(13):4766-9.

  The peroxidase activity of horse cytochrome c was enhanced by its dimerization, where its Compound III (oxy-form) and Compound I (oxoferryl porphyrin π-cation radical) species were detected in theThe peroxidase activity of horse cytochrome c was enhanced by its dimerization, where its Compound III (oxy-form) and Compound I (oxoferryl porphyrin π-cation radical) species were detected in the reactions with hydrogen peroxide and meta-chloroperbenzoic acid, respectively. These results show that oligomeric cytochrome c can contribute as a proapoptotic conformer by the increased peroxidase activity.

• Post-translational His-Cys cross-linkage formation in tyrosinase induced by copper(II)-peroxo species.

  Authors: Nobutaka Fujieda, Takuya Ikeda, Michiaki Murata, Sachiko Yanagisawa, Shigetoshi Aono, Kei Ohkubo, Satoshi Nagao, Takashi Ogura, Shun Hirota, Shunichi Fukuzumi, Yukihiro Nakamura, Yoji Hata, Shinobu Itoh

  Journal of the American Chemical Society. 02/2011; 133(5):1180-3.

  Autocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) underAutocatalytic formation of His-Cys cross-linkage in the enzyme active site of tyrosinase from Aspergillus oryzae has been demonstrated to proceed by the treatment of apoenzyme with Cu(II) under aerobic conditions, where a (μ-η(2):η(2)-peroxo)dicopper(II) species has been suggested to be involved as a key reactive intermediate.

• Supramolecular organization of light-harvesting porphyrin macrorings.

  Authors: Akiharu Satake, Shintaro Azuma, Yusuke Kuramochi, Shun Hirota, Yoshiaki Kobuke

  Chemistry (Weinheim an der Bergstrasse, Germany). 01/2011; 17(3):855-65.

  Porphyrin-based supramolecular nanostructures have been produced by the self-assembly of porphyrin macrorings with three benzoic acid groups (Acid-R) on each side of the rings through cooperativePorphyrin-based supramolecular nanostructures have been produced by the self-assembly of porphyrin macrorings with three benzoic acid groups (Acid-R) on each side of the rings through cooperative carboxyl-carboxyl hydrogen bonds. Structures of the organized Acid-R were analyzed by AFM, and two clear distribution peaks were observed at 3 and 27 nm in the height-distribution histogram. From the overall assessment, the higher objects are considered to be one-dimensional structures standing vertically on the mica substrate. The height corresponds to an 11-mer of a unit Acid-R. Light-harvesting functions were examined by using fluorescence titration, whereby an energy-acceptor molecule (Tripod 2) was employed that strongly interacted with Acid-R units (association constant: 2.0×10(8)  M(-1) ), specifically from the inner pore. The titration results showed that the apparent stoichiometry [Tripod 2]/[Acid-R] was <0.5, and that the value was concentration dependent. Titration results reasonably account for the scheme in which Tripod 2 only interacts with each terminal in the organized Acid-R. The number of organization was fitted to a 10-mer of Acid-R in a 6.8×10(-7)  M solution, and was consistent with that estimated from the AFM results. In the composites of organized Acid-R/Tripod 2, a singlet excitation energy transfer occurred among the Acid-R units, and to Tripod 2. The energy-transfer rate constants were estimated by using the decamer model, which employed kinetic parameters obtained from steady-state and time-resolved fluorescence experiments.

• Efficient reduction of Cys110 thiyl radical by glutathione in human myoglobin.

  Authors: Satoshi Nagao, Osamu Asami, Hiroyuki Yasui, Shun Hirota

  Biochimica et biophysica acta. 01/2011; 1814(4):480-6.

  Human myoglobin (hMb) possesses a cysteine (Cys) residue which is rare among mammalian Mbs. To investigate the effects of this unique Cys residue at the amino acid position 110 (Cys110) on hMbHuman myoglobin (hMb) possesses a cysteine (Cys) residue which is rare among mammalian Mbs. To investigate the effects of this unique Cys residue at the amino acid position 110 (Cys110) on hMb reactions, we studied the reactions of wild type (WT) methMb and its alanine mutant at Cys110 (C110A) with H(2)O(2), particularly in the presence of reduced glutathione (GSH) which is well known as a reducing agent. The formation rates of the ferryloxo (Fe(IV)=O) species by H(2)O(2) under air were about the same for WT and C110A methMbs, whereas the protein decomposed more in the case of WT than C110A hMb. With the addition of GSH, hMb consumed H(2)O(2) faster and decomposition of the protein decreased, where the effects were more prominent in WT than C110A hMb. The radicals produced by the reaction with H(2)O(2) decreased significantly due to the addition of 1mM GSH in the case of WT hMb, but not in the case of C110A hMb. These results show that GSH reduces H(2)O(2)-induced protein decomposition due to reduction of the C110-thiyl radical in WT hMb by electron transfer.

• Crystallization and preliminary X-ray analysis of dimeric and trimeric cytochromes c from horse heart.

  Authors: Midori Taketa, Hirofumi Komori, Yoko Hattori, Satoshi Nagao, Shun Hirota, Yoshiki Higuchi

  Acta crystallographica. Section F, Structural biology and crystallization communications. 11/2010; 66(Pt 11):1477-9.

  Cytochrome c (cyt c) is an electron-transfer protein in the respiratory chain of mitochondria. It is known to form polymers, but its polymerization mechanism is still unknown. Dimeric and trimericCytochrome c (cyt c) is an electron-transfer protein in the respiratory chain of mitochondria. It is known to form polymers, but its polymerization mechanism is still unknown. Dimeric and trimeric cyt c from horse were successfully crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol as a precipitating reagent. The crystal of dimeric cyt c belonged to space group P1, with unit-cell parameters a = 41.8, b = 56.3, c = 60.8 Å, α = 66.3, β = 89.9, γ = 73.7°, whereas that of trimeric cyt c belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.2, b = 95.7, c = 130.9 Å. Initial structure models showed that the crystals of dimeric and trimeric cyt c contained two dimers and two trimers, respectively, in the asymmetric unit.

• Oxoferryl porphyrin/hydrogen peroxide system whose behavior is equivalent to hydroperoxoferric porphyrin.

  Authors: Hiroaki Kitagishi, Mariko Tamaki, Takunori Ueda, Shun Hirota, Takehiro Ohta, Yoshinori Naruta, Koji Kano

  Journal of the American Chemical Society. 11/2010; 132(47):16730-2.

  The reaction between H(2)O(2) and a pyridine-coordinated ferric porphyrin encapsulated by a cyclodextrin dimer yielded a hydroperoxoferric porphyrin intermediate, PFe(III)-OOH, which rapidlyThe reaction between H(2)O(2) and a pyridine-coordinated ferric porphyrin encapsulated by a cyclodextrin dimer yielded a hydroperoxoferric porphyrin intermediate, PFe(III)-OOH, which rapidly decomposed to oxoferryl porphyrin (PFe(IV)═O). Upon reaction with H(2)O(2), PFe(IV)═O reverted to PFe(III)-OOH, which was converted to carbon monoxide-coordinated ferrous porphyrin under a CO atmosphere. PFe(IV)═O in the presence of excess H(2)O(2) behaves as PFe(III)-OOH.

• Cytochrome c polymerization by successive domain swapping at the C-terminal helix.

  Authors: Shun Hirota, Yoko Hattori, Satoshi Nagao, Midori Taketa, Hirofumi Komori, Hironari Kamikubo, Zhonghua Wang, Isao Takahashi, Shigeru Negi, Yukio Sugiura, Mikio Kataoka, Yoshiki Higuchi

  Proceedings of the National Academy of Sciences of the United States of America. 07/2010; 107(29):12854-9.

  Cytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of theCytochrome c (cyt c) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c, the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and (NH(4))(2)HPO(4). The absorption and CD spectra of high-order oligomers (approximately 40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c. For dimeric, trimeric, and tetrameric cyt c, the DeltaH of the oligomer dissociation to monomers was estimated to be about -20 kcal/mol per protomer unit, where Met-heme coordination appears to contribute largely to DeltaH. The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.

• Coherent dynamics and ultrafast excited state relaxation of blue copper protein; plastocyanin.

  Authors: Yutaka Nagasawa, Kenji Fujita, Tetsuro Katayama, Yukihide Ishibashi, Hiroshi Miyasaka, Teruhiro Takabe, Satoshi Nagao, Shun Hirota

  Physical chemistry chemical physics : PCCP. 06/2010; 12(23):6067-75.

  Ultrafast transient absorption measurements in the femtosecond to picosecond time region were carried out for a blue copper protein, plastocyanin (Pc). To compare the dynamical profiles afterUltrafast transient absorption measurements in the femtosecond to picosecond time region were carried out for a blue copper protein, plastocyanin (Pc). To compare the dynamical profiles after photoexcitation upon the ligand-to-metal-charge-transfer (LMCT) band and the d-d transition band, the pump wavelength was set at wavelengths of 597 and 895 nm, respectively. The results were nearly identical, indicating that the transition from the LMCT to the lower ligand field (LF) states takes place in an ultrafast time regime of less than 40 fs. Subsequently, relaxation in the LF state occurs with a time constant of 90 fs and the system returns to the ground state with that of 250 fs. The longest time constant of 1.8 ps was attributed to the vibrational cooling in the ground state. Several wavepacket motions were observed, including Franck-Condon type motion at approximately 510 nm and a Herzberg-Teller type motion at 660-720 nm. Critically damped low-frequency oscillation of approximately 30 cm(-1) was also observed with both excitation wavelengths with the strongest amplitude around 600 nm. This oscillation could be due to the motion of the protein that is ballistically stimulated by ultrafast relaxation.

• Structural basis of the lactate-dependent allosteric regulation of oxygen binding in arthropod hemocyanin.

  Authors: Shun Hirota, Naoki Tanaka, Ivan Micetic, Paolo Di Muro, Satoshi Nagao, Hiroaki Kitagishi, Koji Kano, Richard S Magliozzo, Jack Peisach, Mariano Beltramini, Luigi Bubacco

  The Journal of biological chemistry. 06/2010; 285(25):19338-45.

  Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H(+) and L-lactate. Isothermal titration calorimetric measurements showed thatHemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H(+) and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k(obs) obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.

• Effect of heme modification on oxygen affinity of myoglobin and equilibrium of the acid-alkaline transition in metmyoglobin.

  Authors: Tomokazu Shibata, Satoshi Nagao, Masashi Fukaya, Hulin Tai, Shigenori Nagatomo, Kenji Morihashi, Takashi Matsuo, Shun Hirota, Akihiro Suzuki, Kiyohiro Imai, Yasuhiko Yamamoto

  Journal of the American Chemical Society. 04/2010; 132(17):6091-8.

  Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We haveFunctional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF(3)) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O(2) dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O(2) binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O(2). These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure-function relationship of the protein.

• Reduction of Bis(dithiolene)oxo(disulfido)tungsten(VI) Complex with Dihydrogen Related to the Chemical Function of the Fourth Tungsten-Containing Enzyme (WOR4) from Pyrococcus furiosus.

  Authors: Hideki Sugimoto, Hiroyuki Tano, Kazuo Toyota, Reiko Tajima, Hiroyuki Miyake, Isao Takahashi, Shun Hirota, Shinobu Itoh

  Journal of the American Chemical Society. 12/2009;

  Sulfurization of five-coordinate [W(IV)O(1,2-benzenedithiolate)(2)](2-) proceeds under very mild conditions to form seven-coordinate [W(VI)O(eta(2)-S(2))(1,2-benzenedithiolate)(2)](2-), from whichSulfurization of five-coordinate [W(IV)O(1,2-benzenedithiolate)(2)](2-) proceeds under very mild conditions to form seven-coordinate [W(VI)O(eta(2)-S(2))(1,2-benzenedithiolate)(2)](2-), from which H(2)S was generated by treatment with H(2). This is the first functional model for the fourth tungsten-containing WOR4 enzyme. Kinetics and isotope-scrambling experiments have suggested the existence of six-coordinate W(V)(eta(1)-S(2)) species as a key reaction intermediate.

• Electron transfer from cytochrome c to cupredoxins.

  Authors: Shin-Ichi Takayama, Kiyofumi Irie, Hulin Tai, Takumi Kawahara, Shun Hirota, Teruhiro Takabe, Luis Alcaraz, Antonio Donaire, Yasuhiko Yamamoto

  Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 04/2009;

  Electron transfer (ET) through and between proteins is a fundamental biological process. The activation energy for an ET reaction depends upon the Gibbs energy change upon ET (DeltaG (0)) and theElectron transfer (ET) through and between proteins is a fundamental biological process. The activation energy for an ET reaction depends upon the Gibbs energy change upon ET (DeltaG (0)) and the reorganization energy. Here, we characterized ET from Pseudomonas aeruginosa cytochrome c (551) (PA) and its designed mutants to cupredoxins, Silene pratensis plastocyanin (PC) and Acidithiobacillus ferrooxidans rusticyanin (RC), through measurement of pseudo-first-order ET rate constants (k (obs)). The influence of the DeltaG (0) value for ET from PA to PC or RC on the k (obs) value was examined using a series of designed PA proteins exhibiting a variety of E (m) values, which afford the DeltaG (0) variation range of 58-399 meV. The plots of the k (obs) values obtained against the DeltaG (0) values for both PA-PC and PA-RC redox pairs could be fitted well with a single Marcus equation. We have shown that the ET activity of cytochrome c can be controlled by tuning the E (m) value of the protein through the substitution of amino acid residues located in hydrophobic-core regions relatively far from the redox center. These findings provide novel insights into the molecular design of cytochrome c, which could be utilized for controlling its ET activity by means of protein engineering.

• Modulation of protein-ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3-kinase SH3 domain as model system.

  Authors: Isao Takahashi, Shigeki Kuroiwa, Hanna E Lindfors, Lionel A Ndamba, Yoshitaka Hiruma, Tatsuo Yajima, Nobuyuki Okishio, Marcellus Ubbink, Shun Hirota

  Journal of peptide science : an official publication of the European Peptide Society. 04/2009;

  To photomodulate the interaction of the phosphatidylinositol 3-kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic-1) with a photolabile side chain-to-side chain linker was synthesized.To photomodulate the interaction of the phosphatidylinositol 3-kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic-1) with a photolabile side chain-to-side chain linker was synthesized. The conformation of cyclic-1 differs from that of the parent linear peptide, but becomes identical by UV-irradiation. Accordingly, the binding affinity of cyclic-1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K(d): 3.4 +/- 1.7 and 0.9 +/- 0.3 mM, respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide-protein interactions. Copyright (c) 2009 European Peptide Society and John Wiley & Sons, Ltd.

• Regulating copper-binding affinity with photoisomerizable azobenzene ligand by construction of a self-assembled monolayer.

  Authors: Isao Takahashi, Yuichiro Honda, Shun Hirota

  Angewandte Chemie (International ed. in English). 02/2009; 48(33):6065-8.

• A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating the Access of Water Molecules to the Substrate-Binding Site.

  Authors: Takashi Hayashi, Katsuyoshi Harada, Keisuke Sakurai, Hideo Shimada, Shun Hirota

  Journal of the American Chemical Society. 02/2009;

  Cytochrome P450cam is a heme-containing enzyme which catalyzes hydroxylation of d-camphor. The heme is bound in the heme pocket via noncovalent interactions, where two heme-propionate side chainsCytochrome P450cam is a heme-containing enzyme which catalyzes hydroxylation of d-camphor. The heme is bound in the heme pocket via noncovalent interactions, where two heme-propionate side chains interact with Arg, His, and/or Asp residues. To understand the role of the heme-7-propionate side chain, we prepared reconstituted P450cam with an artificial one-legged heme which has a methyl group at the position of the 7-propionate. Removal of 7-propionate dramatically decreases the d-camphor affinity by 3 orders of magnitude relative to that of the wild-type enzyme, and spectroscopic data indicate that 74% of the ferric P450cam exhibits a low-spin state owing to water molecule occupancy in the substrate-binding site under the normal assay conditions. Thus, the monooxygenase activity of the reconstituted protein is remarkably low due to the decrease in the rate of the first electron transfer from reduced putidaredoxin, whereas 87% of oxidized NADH was utilized to produce 5-hydroxy-d-camphor without any significant uncoupling reactions. X-ray structural analysis of the reconstituted enzyme reveals a novel water array extending from the substrate-binding site to bulk solvent through the position occupied by 7-propionate. This water array appears without causing any major changes in the protein structure with the notable exception of conformational changes occurring at Asp297 and Gln322 residues. We propose that the 7-propionate forms a barrier against entry of bulk water molecules and therefore in combination with Asp297, Arg299, and Gln322 plays an essential role in the process of elimination of the substrate-binding site water cluster which occurs upon d-camphor binding.

• Formation of a bridged butterfly-type mu-eta2:eta2-peroxo dicopper core structure with a carboxylate group.

  Authors: Yasuhiro Funahashi, Tomohide Nishikawa, Yuko Wasada-Tsutsui, Yuji Kajita, Syuhei Yamaguchi, Hidekazu Arii, Tomohiro Ozawa, Koichiro Jitsukawa, Takehiko Tosha, Shun Hirota, Teizo Kitagawa, Hideki Masuda

  Journal of the American Chemical Society. 01/2009; 130(49):16444-5.

• Controlled Production of Amyloid beta Peptide from a Photo-Triggered, Water-Soluble Precursor "Click Peptide"

  Authors: Atsuhiko Taniguchi, Mariusz Skwarczynski, Youhei Sohma, Takuma Okada, Keisuke Ikeda, Halan Prakash, Hidehito Mukai, Yoshio Hayashi, Tooru Kimura, Shun Hirota, Katsumi Matsuzaki, Yoshiaki Kiso

  Chembiochem : a European journal of chemical biology. 12/2008;

  In biological experiments, poor solubility and uncontrolled assembly of amyloid beta peptide (Abeta) 1-42 pose significant obstacles to establish an experiment system that clarifies the function ofIn biological experiments, poor solubility and uncontrolled assembly of amyloid beta peptide (Abeta) 1-42 pose significant obstacles to establish an experiment system that clarifies the function of Abeta1-42 in Alzheimer's disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water-soluble photo-"click peptide" with a coumarin-derived photocleavable protective group that is based on an O-acyl isopeptide method. The click peptide had nearly 100-fold higher water solubility than Abeta1-42 and did not self-assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Abeta1-42. Moreover, the click peptide afforded Abeta1-42 quickly under physiological conditions (pH 7.4, 37 degrees C) by photoirradiation followed by an O-N intramolecular acyl migration. Because the in situ production of intact Abeta1-42 from the click peptide could improve the difficulties in handling Abeta1-42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Abeta1-42 in AD.

• Formation of a Bridged Butterfly-Type mu-eta(2):eta(2)-Peroxo Dicopper Core Structure with a Carboxylate Group.

  Authors: Yasuhiro Funahashi, Tomohide Nishikawa, Yuko Wasada-Tsutsui, Yuji Kajita, Syuhei Yamaguchi, Hidekazu Arii, Tomohiro Ozawa, Koichiro Jitsukawa, Takehiko Tosha, Shun Hirota, Teizo Kitagawa, Hideki Masuda

  Journal of the American Chemical Society. 12/2008;