KINDAI UNIVERSITY


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SAKURAI Kazumasa

Profile

FacultyInstitute of Advanced Technology / Graduate School of Biology-Oriented Science and Technology
PositionAssociate Professor
Degree
Commentator Guidehttps://www.kindai.ac.jp/meikan/310-sakurai-kazumasa.html
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Last Updated :2020/09/30

Education and Career

Education

  •  - 2002 , Osaka University, graduate school of science
  •  - 2000 , Osaka University, School of Science
  •   1996 04  - 2000 03 , Osaka University, School of Science

Academic & Professional Experience

  •   2005 04 ,  - 2013 03 , Assist. Prof., Inst. Protein Res., Osaka Univ.

Research Activities

Research Interests

  • amyloid, protein, protein folding, NMR, biophysics, Dynamics

Published Papers

  • Isoelectric point-amyloid formation of α-synuclein extends the generality of the solubility and supersaturation-limited mechanism, Koki Furukawa, Cesar Aguirre, Masatomo So, Kenji Sasahara, Yohei Miyanoiri, Kazumasa Sakurai, Keiichi Yamaguchi, Kensuke Ikenaka, Hideki Mochizuki, Jozsef Kardos, Yasushi Kawata, Yuji Goto, Current Research in Structural Biology, Current Research in Structural Biology, 2, 35 - 44, Apr. 05 2020 , Refereed
  • Heating during agitation of β2-microglobulin reveals that supersaturation breakdown is required for amyloid fibril formation at neutral pH., Noji M, Sasahara K, Yamaguchi K, So M, Sakurai K, Kardos J, Naiki H, Goto Y, The Journal of biological chemistry, The Journal of biological chemistry, in press, Sep. 2019 , Refereed
  • Loosening of side-chain packing associated with perturbations in peripheral dynamics induced by the D76N mutation of β2-microglobulin revealed by pressure-NMR and molecular dynamic simulations, Sakurai K, Tomiyama R, Shiraki T, Yonezawa Y, Biomolecules, Biomolecules, 9(9), 491, Sep. 2019 , Refereed
  • Conformational Properties Relevant to the Amyloidogenicity of beta2-Microglobulin Analyzed Using Pressure- and Salt-Dependent Chemical Shift Data., Sakurai K, Maeno A, Lee YH, Akasaka K, The Journal of Physical Chemistry B, The Journal of Physical Chemistry B, 123(4), 836 - 844, Jan. 2019 , Refereed
  • Non-Native α-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the β-Barrel in β-Lactoglobulin., Sakurai K, Yagi M, Konuma T, Takahashi S, Nishimura C, Goto Y, Biochemistry, Biochemistry, 56(36), 4799 - 4807, Sep. 2017 , Refereed
  • Non-Native alpha-Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the beta-Barrel in beta-Lactoglobulin, Sakurai, K, Yagi, M, Konuma, T, Takahashi, S, Nishimura, C, Goto, Y, Biochemistry, Biochemistry, Aug. 2017 , Refereed
  • Highly Collapsed Conformation of the Initial Folding Intermediates of beta-Lactoglobulin with Non-Native alpha-Helix, Tsuyoshi Konuma, Kazumasa Sakurai, Masanori Yagi, Yuji Goto, Tetsuro Fujisawa, Satoshi Takahashi, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 427(19), 3158 - 3165, Sep. 2015 , Refereed
    Summary:In the folding of beta-lactoglobulin (beta LG), a predominantly beta-sheet protein, a transient intermediate possessing an excess amount of non-native a-helix is formed within a few milliseconds. To characterize the early folding dynamics of beta LG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native alpha-helix formation within 200 mu s. Time-resolved SAXS showed that the radius of gyration (R-g) of the intermediate at 309 mu s was 23.3 +/- 0.7 angstrom, indicating a considerable collapse from the unfolded state having R-g of 35.1 +/- 7.1 angstrom. Further compaction to R-g of 21.2 +/- 0.3 angstrom occurred with a time constant of 28 +/- 11 ms. Pair distribution functions showed that the intermediate at 300 mu s comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of alpha-helix but similar R-g of 23.0 +/- 0.8 angstrom, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native alpha-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of beta LG. (C) 2015 Elsevier Ltd. All rights reserved.
  • Effects of a reduced disulfide bond on aggregation properties of the human IgG1 CH3 domain., Sakurai K, Nakahata R, Lee YH, Kardos J, Ikegami T, Goto Y, Biochimica et biophysica acta, Biochimica et biophysica acta, Mar. 2015 , Refereed
  • A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication., Kitayama H, Yoshimura Y, So M, Sakurai K, Yagi H, Goto Y, Biochimica et biophysica acta, Biochimica et biophysica acta, 1834(12), 2640 - 2646, Dec. 2013 , Refereed
  • The monomer-seed interaction mechanism in the formation of the β2-microglobulin amyloid fibril clarified by solution NMR techniques., Yanagi K, Sakurai K, Yoshimura Y, Konuma T, Lee YH, Sugase K, Ikegami T, Naiki H, Goto Y, J Mol Biol, J Mol Biol, 422(3), 390 - 402, Sep. 2012 , Refereed
  • The Monomer-Seed Interaction Mechanism in the Formation of the beta 2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques, Kotaro Yanagi, Kazumasa Sakurai, Yuichi Yoshimura, Tsuyoshi Konuma, Young-Ho Lee, Kenji Sugase, Takahisa Ikegami, Hironobu Naiki, Yuji Goto, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 422(3), 390 - 402, Sep. 2012 , Refereed
    Summary:Amyloid fibrils are proteinous aggregates associated with various diseases, including Alzheimer's disease, type II diabetes, and dialysis-related amyloidosis. It is generally thought that, during the progression of these diseases, a precursor peptide or protein assumes a partially denatured structure, which interacts with the fibril seed to change into the final amyloid form. beta 2-Microglobulin (beta 2m), associated with dialysis-related amyloidosis, is known to form amyloid fibrils at low pH via a partially structured state. However, the molecular mechanism by which the conformation of beta 2m changes from the precursor to the final fibril structure is poorly understood. We performed various NMR experiments to characterize acid-denatured beta 2m. The analysis of the transverse relaxation rates revealed that acid-denatured beta 2m undergoes a structural exchange with an extensively unfolded form. The results of transferred cross-saturation experiments indicated that residues with a residual structure in the acid-denatured state are associated with the interaction with the fibril seed. Our experimental data suggest the partially structured state to be "activated" to become extensively unfolded, in which state the hydrophobic residues are exposed and associate with the seed. Our results provide general information about the extension of amyloid fibrils. (c) 2012 Elsevier Ltd. All rights reserved.
  • A Circumventing Role for the Non-Native Intermediate in the Folding of beta-Lactoglobulin, Kazumasa Sakurai, Shunsuke Fujioka, Tsuyoshi Konuma, Masanori Yagi, Yuji Goto, BIOCHEMISTRY, BIOCHEMISTRY, 50(29), 6498 - 6507, Jul. 2011 , Refereed
    Summary:Folding experiments have suggested that some proteins have kinetic intermediates with a non-native structure. A simple G (o) over bar model does not explain such non-native intermediates. Therefore, the folding energy landscape of proteins with non-native intermediates should have characteristic properties. To identify such properties, we investigated the folding of bovine beta-lactoglobulin (beta LG). This protein has an intermediate with a non-native alpha-helical structure, although its native form is predominantly composed of beta-structure. In this study, we prepared mutants whose a-helical and beta-sheet propensities are modified and observed their folding using a stopped-flow circular dichroism apparatus. One interesting finding was that E44L, whose beta-sheet propensity was increased, showed a folding intermediate with an amount of beta-structure similar to that of the wild type, though its folding took longer. Thus, the intermediate seems to be a trapped intermediate. The high a-helical propensity of the wild-type sequence likely causes the folding pathway to circumvent such time-consuming intermediates. We propose that the role of the non-native intermediate is to control the pathway at the beginning of the folding reaction.
  • Kinetic Intermediates of beta(2)-Microglobulin Fibril Elongation Probed by Pulse-Labeling H/D Exchange Combined with NMR Analysis, Tsuyoshi Konuma, Eri Chatani, Masanori Yagi, Kazumasa Sakurai, Takahisa Ikegami, Hironobu Naiki, Yuji Goto, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 405(3), 851 - 862, Jan. 2011 , Refereed
    Summary:Amyloid fibril elongation in denatured proteins involves cycles of coupled binding and misfolding. To gain insights into possible kinetic intermediates, we performed hydrogen/deuterium exchange of amide protons during fibril elongation with beta(2)-microglobulin (beta(2)-m) at pD = 2.5, under which conditions beta(2)-m is acid denatured. To study the conformational change in monomeric beta(2)-m monitored by NMR spectroscopy, we used N-15-labeled monomers and nonlabeled seeds. Pulse-labeling hydrogen/deuterium exchange with a quenched-flow apparatus indicated that the rate-limiting intermediate at pD = 2.5 is not protected from the exchange, even disrupting a hydrophobic cluster present in the acid-denatured beta(2)-m. Significant protection was acquired upon transition to the fibrils. In view of the suggestion that the rate-limiting intermediates are bound to the lateral surface of seed fibrils, weak interactions with a largely unfolded conformation might be useful for their dynamic sliding to the growing ends. The results support a new model of fibril elongation with intermediates bound to the lateral surface of seeds. (C) 2010 Elsevier Ltd. All rights reserved.
  • Structural dynamics and folding of beta-lactoglobulin probed by heteronuclear NMR., Sakurai K, Konuma T, Yagi M, Goto Y, Biochimica et biophysica acta, Biochimica et biophysica acta, 1790(6):527-37(6), 527 - 537, Jun. 2009 , Refereed
  • Principal component analysis of the pH-dependent conformational transitions of bovine beta-lactoglobulin monitored by heteronuclear NMR, Kazumasa Sakurai, Yuji Goto, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(39), 15346 - 15351, Sep. 2007 , Refereed
    Summary:To clarify the pH-dependent conformational transitions of proteins, we propose an approach in which structural changes monitored by heteronuclear sequential quantum correlation (HSQC) spectroscopy were analyzed by using a principal component analysis (PCA). We use bovine P-lactoglobulin, a protein widely used in protein folding studies, as a target. First, we measured HSQC spectra at various pH values and subjected them to a PCA. The analysis revealed three apparent transitions with pK(a) values of 2.9, 4.9, and 6.8, consistent with previous reports using different methods. Next, Gdn-HCl-induced unfolding was examined by measuring tryptophan fluorescence at various pH values. Between pH 2 and 8, beta-lactoglobulin exhibited a number of structural transitions as well as changes in stability represented by the free energy change of unfolding, Delta G(U). By combining the NMR and fluorescence results, the change in Delta G(U) was suggested to result from the decreased pKa of some acidic residues. Notably, the native state at neutral pH is destabilized by deprotonation of Glu-89, leading to an increase in the relative population of the intermediate. Thus, the PCA of pH-dependent HSQC spectra provides a more comprehensive understanding of the stability and function of proteins.
  • Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3, K Sakurai, M Oobatake, Y Goto, PROTEIN SCIENCE, PROTEIN SCIENCE, 10(11), 2325 - 2335, Nov. 2001 , Refereed
    Summary:Although bovine beta -lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO4 was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaCIO4, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta -lactoglobutin at pH 3.0 with NaClO4. The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degreesC and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaCIO4 the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of P-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.
  • Conformation and stability of thiol-modified bovine beta-lactoglobulin, K Sakai, K Sakurai, M Sakai, M Hoshino, Y Goto, PROTEIN SCIENCE, PROTEIN SCIENCE, 9(9), 1719 - 1729, Sep. 2000 , Refereed
    Summary:Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. beta-Lactoglobulin A has a free thiol at Cysl21, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCl at pH 7.5, producing a modified beta-lactoglobulin (TNB-blg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-blg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional H-1-NMR. The CD spectra of TNB-blg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the ct-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-blg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-blg is monomeric while the intact protein is dimeric. Tn contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-blg were monomeric. The unfolding transition of TNB-blg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The H-1-NMR spectrum for TNB-blg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-blg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-blg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.
  • Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation, Ayame Nitani, Hiroya Muta, Masayuki Adachi, Masatomo So, Kenji Sasahara, Kazumasa Sakurai, Eri Chatani, Kazumitsu Naoe, Hirotsugu Ogi, Damien Hall, Yuji Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 292(52), 21219 - 21230, Dec. 2017 , Refereed
    Summary:Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 degrees C. At concentrations lower than 20 g/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.
  • Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies, Satomi Inaba, Akihiro Maeno, Kazumasa Sakurai, Sunilkumar Puthenpurackal Narayanan, Takahisa Ikegami, Kazuyuki Akasaka, Masayuki Oda, FEBS JOURNAL, FEBS JOURNAL, 282(23), 4497 - 4514, Dec. 2015 , Refereed
    Summary:The conformational fluctuation in the minimum DNA-binding domain of cMyb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30-70 degrees C, with a transition temperature of approximately 50 degrees C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both 1 H one-dimensional and N-15/H-1 two-dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild-type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity- filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild-type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37 degrees C and is likely beneficial for its biological function: DNA-binding. This result is in agreement with the concept of an excited-state conformer that exists in equilibrium with the dominant ground-state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide-type c-Myb DNA-binding domain to produce an appropriate fraction of the locally fluctuating state at 37 degrees C, which is more amenable to DNA-binding.
  • Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies, Satomi Inaba, Akihiro Maeno, Kazumasa Sakurai, Sunilkumar Puthenpurackal Narayanan, Takahisa Ikegami, Kazuyuki Akasaka, Masayuki Oda, FEBS Journal, FEBS Journal, 282(23), 4497 - 4514, Dec. 01 2015 , Refereed
    Summary:The conformational fluctuation in the minimum DNA-binding domain of c-Myb, repeats 2 and 3 (R2R3), was studied under closely physiological conditions. A global unfolding transition, involving both the main chain and the side chains, was found to take place at the approximate temperature range 30-70 C, with a transition temperature of approximately 50 C. In addition, the observation of simultaneous shift change and broadening of NMR signals in both 1H one-dimensional and 15N/1H two-dimensional NMR spectra indicated the occurrence of locally fluctuating state at physiological temperature. In the wild-type protein containing a cavity in R2, the local fluctuation of R2 is more prominent than that of R3, whereas it is suppressed in the cavity-filled mutant, V103L. This indicates that the cavity in R2 contributes significantly to the conformational instability and the transition into the locally fluctuating state. For the wild-type R2R3 protein, the more dynamic conformer is estimated to be present to some extent at 37 C and is likely beneficial for its biological function: DNA-binding. This result is in agreement with the concept of an excited-state conformer that exists in equilibrium with the dominant ground-state conformer and acts as the functional conformer of the protein. From the findings of the present study, it appears that the tandem repeats of two small domains with no disulfide bonds and with a destabilizing cavity function as the evolutionary strategy of the wide-type c-Myb DNA-binding domain to produce an appropriate fraction of the locally fluctuating state at 37 C, which is more amenable to DNA-binding. Database Chemical shifts and peak lists have been deposited in the Biological Magnetic Resonance Bank under entries 11584 and 11585.
  • Effects of a reduced disulfide bond on aggregation properties of the human IgG1 C(H)3 domain, Kazumasa Sakurai, Ryosuke Nakahata, Young-Ho Lee, Jozsef Kardos, Takahisa Ikegami, Yuji Goto, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1854(10), 1526 - 1535, Oct. 2015 , Refereed
    Summary:Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. An IgG1 molecule, which is now mainly used for antibody preparation, consists of a total of 12 immunoglobulin domains. Each domain has one disulfide bond. The C(H)3 domain is the C-terminal domain of the heavy chain of IgG1. The disulfide bonds of some of the C(H)3 domains are known to be reduced in recombinant human monoclonal antibodies. The lack of intramolecular disulfide bonds may decrease the stability and increase the aggregation propensity of an antibody molecule. To investigate the effects of a reduced disulfide bond in the C(H)3 domain on conformational stability and aggregation propensity, we performed several physicochemical measurements including circular dichroism, differential scanning calorimetty (DSC), and 2D NMR. DSC measurements showed that both the stability and reversibility of the reduced form were lower than those of the oxidized form. In addition, detailed analyses of the thermal denaturation data revealed that, although a dominant fraction of the reduced form retained a stable dimeric structure, some fractions assumed a less-specifically associated oligomeric state between monomers. The results of the present study revealed the characteristic aggregation properties of antibody molecules. (C) 2015 Elsevier B.V. All rights reserved.
  • Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation, Masayuki Adachi, Masatomo So, Kazumasa Sakurai, Jozsef Kardos, Yuji Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 290(29), 18134 - 18145, Jul. 2015 , Refereed
    Summary:Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used beta(2)-microglobulin (beta 2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. Whenultrasonication was used to accelerate the spontaneous fibrillation of beta 2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained beta-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of beta 2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.
  • A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication, Hiroki Kitayama, Yuichi Yoshimura, Masatomo So, Kazumasa Sakurai, Hisashi Yagi, Yuji Goto, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1834(12), 2640 - 2646, Dec. 2013 , Refereed
    Summary:Protein crystals form in supersaturated solutions via a nudeation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils. (C) 2013 Elsevier B.V. All rights reserved.
  • Principal component analysis of chemical shift perturbation data of a multiple-ligand-binding system for elucidation of respective binding mechanism, Tsuyoshi Konuma, Young-Ho Lee, Yuji Goto, Kazumasa Sakurai, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 81(1), 107 - 118, Jan. 2013 , Refereed
    Summary:Chemical shift perturbations (CSPs) in NMR spectra provide useful information about the interaction of a protein with its ligands. However, in a multiple-ligand-binding system, determining quantitative parameters such as a dissociation constant (Kd) is difficult. Here, we used a method we named CS-PCA, a principal component analysis (PCA) of chemical shift (CS) data, to analyze the interaction between bovine beta-lactoglobulin (beta LG) and 1-anilinonaphthalene-8-sulfonate (ANS), which is a multiple-ligand-binding system. The CSP on the binding of ANS involved contributions from two distinct binding sites. PCA of the titration data successfully separated the CSP pattern into contributions from each site. Docking simulations based on the separated CSP patterns provided the structures of beta LGANS complexes for each binding site. In addition, we determined the Kd values as 3.42 x 10-4M2 and 2.51 x 10-3M for Sites 1 and 2, respectively. In contrast, it was difficult to obtain reliable Kd values for respective sites from the isothermal titration calorimetry experiments. Two ANS molecules were found to bind at Site 1 simultaneously, suggesting that the binding occurs cooperatively with a partial unfolding of the beta LG structure. On the other hand, the binding of ANS to Site 2 was a simple attachment without a significant conformational change. From the present results, CS-PCA was confirmed to provide not only the positions and the Kd values of binding sites but also information about the binding mechanism. Thus, it is anticipated to be a general method to investigate proteinligand interactions. Proteins 2013. (c) 2012 Wiley Periodicals, Inc.
  • Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation, Yuichi Yoshimura, Yuxi Lin, Hisashi Yagi, Young-Ho Lee, Hiroki Kitayama, Kazumasa Sakurai, Masatomo So, Hirotsugu Ogi, Hironobu Naiki, Yuji Goto, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 109(36), 14446 - 14451, Sep. 2012 , Refereed
    Summary:Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two\ forms are often treated indiscriminately. beta(2)-microglobulin (beta 2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.
  • A Back Hydrogen Exchange Procedure via the Acid-Unfolded State for a Large Protein, Mototaka Suzuki, Kazumasa Sakurai, Young-Ho Lee, Takahisa Ikegami, Keiichi Yokoyama, Yuji Goto, BIOCHEMISTRY, BIOCHEMISTRY, 51(28), 5564 - 5570, Jul. 2012 , Refereed
    Summary:A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (H2O)-H-2 should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry 50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (H2O)-H-2, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements.
  • Binding Energetics of Ferredoxin-NADP(+) Reductase with Ferredoxin and Its Relation to Function, Young-Ho Lee, Takahisa Ikegami, Daron M. Standley, Kazumasa Sakurai, Toshiharu Hase, Yuji Goto, CHEMBIOCHEM, CHEMBIOCHEM, 12(13), 2062 - 2070, Sep. 2011 , Refereed
    Summary:To obtain insight into the motional features of proteins for enzymatic function, we studied binding reactions between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd) using isothermal titration calorimetry and NMR-based magnetic relaxation and hydrogen/deuterium exchange (HDex). Fd-FNR binding was accompanied by endothermic reactions and driven by the entropy gain. Component-wise analysis of the net entropy change revealed that increases in the conformational entropy of the Fd-FNR complex contributed largely to stabilizing the complex. Intriguingly, analyses of magnetic relaxation and HDex rates with X-ray B factor implied that Fd binding led to both structural stiffening and softening of FNR. Enhanced FNR backbone fluctuations suggest favorable contributions to the net conformational entropy. Fd-bound FNR further showed that relatively large-scale motions of the C terminus, a gatekeeper for interactions of NADP(+)(H), were quenched in the closed form, thereby facilitating exit of NADP(+)(H). This can provide a first dynamic structure-based explanation for the negative cooperativity between Fd and NADP(+)(H) via FNR.
  • Ultrasonication-Dependent Acceleration of Amyloid Fibril Formation, Masatomo So, Hisashi Yagi, Kazumasa Sakurai, Hirotsugu Ogi, Hironobu Naiki, Yuji Goto, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 412(4), 568 - 577, Sep. 2011 , Refereed
    Summary:Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With beta(2)-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins. (C) 2011 Elsevier Ltd. All rights reserved.
  • Hexafluoroisopropanol Induces Amyloid Fibrils of Islet Amyloid Polypeptide by Enhancing Both Hydrophobic and Electrostatic Interactions, Kotaro Yanagi, Mizue Ashizaki, Hisashi Yagi, Kazumasa Sakurai, Young-Ho Lee, Yuji Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 286(27), 23959 - 23966, Jul. 2011 , Refereed
    Summary:Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the alpha-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.
  • Inhibition of beta(2)-Microglobulin Amyloid Fibril Formation by alpha(2)-Macroglobulin, Daisaku Ozawa, Kazuhiro Hasegawa, Young-Ho Lee, Kazumasa Sakurai, Kotaro Yanagi, Tadakazu Ookoshi, Yuji Goto, Hironobu Naiki, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 286(11), 9668 - 9676, Mar. 2011 , Refereed
    Summary:The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, alpha(2)-macroglobulin (alpha 2M), an extracellular chaperone, forms a complex with beta(2)-microglobulin (beta 2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that alpha 2M substoichiometrically inhibited the beta 2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between alpha 2M and beta 2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric alpha 2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric alpha 2M interacted with SDS-denatured beta 2-m. At a physiologically relevant acidic pH and in the presence of heparin, alpha 2M was also dissociated into dimers, and both tetrameric and dimeric alpha 2M interacted with beta 2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native beta 2-m is denatured, tetrameric alpha 2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured beta 2-m, thus dimeric alpha 2M as well as tetrameric alpha 2M may play an important role in controlling beta 2-m amyloid fibril formation.
  • Destruction of Amyloid Fibrils of Keratoepithelin Peptides by Laser Irradiation Coupled with Amyloid-specific Thioflavin, Daisaku Ozawa, Yuichi Kaji, Hisashi Yagi, Kazumasa Sakurai, Toru Kawakami, Hironobu Naiki, Yuji Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 286(12), 10856 - 10863, Mar. 2011 , Refereed
    Summary:Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments and amyloid beta by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.
  • Kinetic intermediates of β2-microglobulin fibril elongation probed by pulse-labeling H/D exchange combined with NMR analysis, Tsuyoshi Konuma, Eri Chatani, Masanori Yagi, Kazumasa Sakurai, Takahisa Ikegami, Hironobu Naiki, Yuji Goto, Journal of Molecular Biology, Journal of Molecular Biology, 405(3), 851 - 862, Jan. 21 2011
    Summary:Amyloid fibril elongation in denatured proteins involves cycles of coupled binding and misfolding. To gain insights into possible kinetic intermediates, we performed hydrogen/deuterium exchange of amide protons during fibril elongation with β2-microglobulin (β2-m) at pD = 2.5, under which conditions β2-m is acid denatured. To study the conformational change in monomeric β2-m monitored by NMR spectroscopy, we used 15N-labeled monomers and nonlabeled seeds. Pulse-labeling hydrogen/deuterium exchange with a quenched-flow apparatus indicated that the rate-limiting intermediate at pD = 2.5 is not protected from the exchange, even disrupting a hydrophobic cluster present in the acid-denatured β2-m. Significant protection was acquired upon transition to the fibrils. In view of the suggestion that the rate-limiting intermediates are bound to the lateral surface of seed fibrils, weak interactions with a largely unfolded conformation might be useful for their dynamic sliding to the growing ends. The results support a new model of fibril elongation with intermediates bound to the lateral surface of seeds. © 2010 Elsevier Ltd. All rights reserved.
  • Direct observation of minimum-sized amyloid fibrils using solution NMR spectroscopy, Yuichi Yoshimura, Kazumasa Sakurai, Young-Ho Lee, Takahisa Ikegami, Eri Chatani, Hironobu Naiki, Yuji Goto, PROTEIN SCIENCE, PROTEIN SCIENCE, 19(12), 2347 - 2355, Dec. 2010 , Refereed
    Summary:It is challenging to investigate the structure and dynamics of amyloid fibrils at the residue and atomic resolution because of their high molecular weight and heterogeneous properties Here, we used solution nuclear magnetic resonance (NMR) spectroscopy to characterize the conformation and flexibility of amyloid fibrils of beta(2)-microglobulin (beta 2m), for which direct observation of solution NMR could not be made Ultrasonication led to fragmentation producing a solution of minimum-sized fibrils with a molecular weight of around 6 MDa In (1)H-(15)N heteronuclear single-quantum correlation measurements, five signals, derived from N-terminal residues (i e, Ile1, Gln2, Arg3, Thr4, and Lys6), were newly detected Signal strength decreased with the distance from the N-terminal end Capping experiments with the unlabeled beta 2m monomer indicated that the signals originated from molecules located inside the fibrils Ultrasonication makes the residues with moderate flexibility observable by reducing size of the fibrils Thus, solution NMR measurements of ultrasonicated fibrils will be promising for studying the structure and dynamics of fibrils
  • Pre-Steady-State Kinetic Analysis of the Elongation of Amyloid Fibrils of beta(2)-Microglobulin with Tryptophan Mutagenesis, Eri Chatani, Reina Ohnishi, Tsuyoshi Konuma, Kazumasa Sakurai, Hironobu Naiki, Yuji Goto, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 400(5), 1057 - 1066, Jul. 2010 , Refereed
    Summary:Amyloid fibrils elongate seed dependently, with preformed fibrils providing a template for propagation of amyloidogenic conformation. Most seeding experiments use relatively few seed fibrils in comparison with monomers, resembling steady-state enzyme kinetics. Pre-steady-state kinetics should also be useful for characterizing the elongation process. With beta(2)-microglobulin (beta(2)-m), a protein responsible for dialysis-related amyloidosis, we measured the pre-steady-state kinetics of fibril elongation at pH 2.5, conditions under which the monomer is largely unfolded. beta(2)-m has Trp residues at positions 60 and 95. We used three single Trp mutants and fluorescence spectroscopy to study structural change upon fibril elongation. To focus on conformational change in monomers, we prepared seeds with a mutant without a Trp residue. At a fixed concentration of monomeric beta(2)-m, the apparent rate of fibril elongation increased with an increase in the concentration of seeds and then saturated, suggesting the accumulation of a rate-limiting intermediate. Importantly, saturation occurred at a seed/monomer ratio of around 10, as expressed by the concentration of the monomer. Because the number of monomers constituting the seed fibrils is much larger than 10, the results suggest that the elongation process is limited by "non-active-site binding." Spectral analysis indicated that, upon this non-active-site binding, both Trp60 and Trp95 are exposed to the solvent, and then only Trp60 is buried upon transition to the fibrils. We propose a new model of fibril elongation in which non-active-site binding plays a major role. (C) 2010 Elsevier Ltd. All rights reserved
  • Laser-induced Propagation and Destruction of Amyloid beta Fibrils, Hisashi Yagi, Daisaku Ozawa, Kazumasa Sakurai, Toru Kawakami, Hiroki Kuyama, Osamu Nishimura, Toshinori Shimanouchi, Ryoichi Kuboi, Hironobu Naiki, Yuji Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 285(25), 19660 - 19667, Jun. 2010 , Refereed
    Summary:The amyloid deposition of amyloid beta (A beta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta 2-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on A beta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed A beta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.
  • NMR-based characterization of a refolding intermediate of beta 2-microglobulin labeled using a wheat germ cell-free system, Atsushi Kameda, Eugene-Hayato Morita, Kazumasa Sakurai, Hironobu Naiki, Yuji Goto, PROTEIN SCIENCE, PROTEIN SCIENCE, 18(8), 1592 - 1601, Aug. 2009 , Refereed
    Summary:In patients with dialysis-related amyloidosis, beta 2-microglobulin (beta 2-m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of beta 2-m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non-native trans-Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell-free protein synthesis and NMR techniques. The HSQC spectra of beta 2-ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the beta-sheet regions especially the last half of the beta B strand and the first half of the beta E strand, both suggested to be important for amyloidogenicity, may transform beta 2-m into an amyloidogenic structure.
  • Kinetic Coupling of Folding and Prolyl Isomerization of beta(2)-Microglobulin Studied by Mutational Analysis, Michiko Sakata, Eri Chatani, Atsushi Kameda, Kazumasa Sakurai, Hironobu Naiki, Yuji Goto, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 382(5), 1242 - 1255, Oct. 2008 , Refereed
    Summary:beta(2)-Microglobulin (beta 2-m), a protein responsible for dialysis-related amyloidosis, adopts a typical immunoglobulin domain fold with the N-terminal peptide bond of Pro32 in a cis isomer. The refolding of beta 2-m is limited by the slow trans-to-cis isomerization of Pro32, implying that intermediates with a non-native trans-Pro32 isomer are precursors for the formation of amyloid fibrils. To obtain further insight into the Pro-limited folding of beta 2-m, we Studied the Gdn-HCl-dependent unfolding/refolding kinetics using two mutants (W39 and P32V beta 2-ms) as well as the wild-type beta 2-m. W39 beta 2-m is a triple mutant in which both of the authentic Trip residues (Trp60 and Trp95) are replaced by Phe and a buried Trp common to other immunoglobulin domains is introduced at the position of Leu39 (i.e., L39W/W60F/W95F). W39 Q-m exhibits a dramatic quenching of fluorescence upon folding, enabling a detailed analysis of Pro-limited unfolding/refolding. On the other hand, P32V beta 2-m is a mutant in which Pro32 is replaced by Val, useful for probing the kinetic role of the trans-to-cis isomerization of Pro32. A comparative analysis of the unfolding/refolding kinetics of these Mutants including three types of double-jump experiments revealed the prolyl isomerization to be coupled with the conformational transitions, leading to apparently unusual kinetics, particularly for the unfolding. We suggest that careful consideration of the kinetic Coupling Of unfolding/refolding and prolyl isomerization which has tended to be neglected in recent Studies, is essential for clarifying the mechanism of protein folding and, moreover, its biological significance. (C) 2008 Elsevier Ltd. All rights reserved.
  • Disulfide-linked bovine beta-lactoglobulin dimers fold slowly, navigating a glassy folding landscape, Masanori Yagi, Atsushi Kameda, Kazumasa Sakurai, Chiaki Nishimura, Yuji Goto, BIOCHEMISTRY, BIOCHEMISTRY, 47(22), 5996 - 6006, Jun. 2008 , Refereed
    Summary:To gain insight into the folding of large proteins, we constructed a bovine beta-lactoglobulin (beta-lg) dimeric mutant, A34C/C121A beta-lg. In the mutant, a free thiol group of wild-type beta-lg at Cys121 was removed and two beta-lg molecules were linked by a disulfide bridge through Cys34 created at the dimer's interface. Under strongly native conditions at low concentrations of urea, the refolding yield of A34C/C121A beta-lg was low when monitored by heteronuclear NMR spectroscopy. However, under marginally native conditions, the yield improved notably, although the refolding was still slow. H-D exchange pulse labeling monitored using heteronuclear NMR spectroscopy indicated that A34C/C121A beta-lg forms a folding intermediate similar to monomeric C121A beta-lg in spite of its slow folding. These results indicate that the rapid formation of folding intermediates driven by local interactions occurs in a manner independent of the molecular size and that, if the non-native interactions are too strong, the kinetic trap is set, leading to a glasslike misfolded state. The results suggest the important roles of marginal stability and pathways in making the folding of large proteins possible.
  • Principal component analysis of the pH-dependent conformational transitions of bovine beta-lactoglobulin monitored by heteronuclear NMR, Kazumasa Sakurai, Yuji Goto, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104(39), 15346 - 15351, Sep. 2007
    Summary:To clarify the pH-dependent conformational transitions of proteins, we propose an approach in which structural changes monitored by heteronuclear sequential quantum correlation (HSQC) spectroscopy were analyzed by using a principal component analysis (PCA). We use bovine P-lactoglobulin, a protein widely used in protein folding studies, as a target. First, we measured HSQC spectra at various pH values and subjected them to a PCA. The analysis revealed three apparent transitions with pK(a) values of 2.9, 4.9, and 6.8, consistent with previous reports using different methods. Next, Gdn-HCl-induced unfolding was examined by measuring tryptophan fluorescence at various pH values. Between pH 2 and 8, beta-lactoglobulin exhibited a number of structural transitions as well as changes in stability represented by the free energy change of unfolding, Delta G(U). By combining the NMR and fluorescence results, the change in Delta G(U) was suggested to result from the decreased pKa of some acidic residues. Notably, the native state at neutral pH is destabilized by deprotonation of Glu-89, leading to an increase in the relative population of the intermediate. Thus, the PCA of pH-dependent HSQC spectra provides a more comprehensive understanding of the stability and function of proteins.
  • High-resolution crystal structure of beta(2)-microglobulin formed at pH 7.0, Kentaro Iwata, Takanori Matsuura, Kazumasa Sakurai, Atsushi Nakagawa, Yuji Goto, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 142(3), 413 - 419, Sep. 2007 , Refereed
    Summary:beta(2)-Microglobulin (beta 2-m), a light chain of the major histocompatibility complex class 1, forms amyloid fibrils in patients undergoing long-term haemodialysis, causing dialysis-related amyloidosis. Based on a comparison of the X-ray structure obtained at pH 5.7 and that of beta 2-m in the histocompatibility complex, it has been proposed that the continuous D-strand observed in the crystal structure at pH 5.7 increases the propensity of beta 2-m to self-associate via edge-to-edge interactions, thus initiating the formation of fibrils. To obtain further insight into the mechanism by which amyloid fibrils form, we determined the crystal structure of beta 2-m at pH 7.0 at a resolution of up to 1.13 angstrom. The crystal structure at pH 7.0 was basically the same as that at pH 5.6, suggesting that the conversion of the P-bulge in strand D into a contiguous P-strand is not unique to the crystals formed under slightly acidic conditions. In other words, although the formation of beta 2-m fibrils was enhanced under acidic conditions, it remains unknown if it is related to the increased propensity for the disappearance of the P-bulge in strand D. We consider that the enhanced fibrillation is more directly coupled with the decreased stability leading to the increased propensity of exposing amyloidogenic regions.
  • Promiscuous binding of ligands by beta-lactoglobulin involves hydrophobic interactions and plasticity, Tsuyoshi Konuma, Kazumasa Sakurai, Yuji Goto, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 368(1), 209 - 218, Apr. 2007 , Refereed
    Summary:Bovine-lactoglobulin (beta LG) binds a variety of hydrophobic ligands, though precisely how is not clear. To understand the structural basis of this promiscuous binding, we studied the-interaction of beta LG with palmitic acid (PA) using heteronuclear NMR spectroscopy. The titration was monitored using tryptophan fluorescence and a HSQC spectrum confirmed a 1:1 stoichiometry for the PA-beta LG complex. Upon the binding of PA, signal disappearances and large changes in chemical shifts were observed for the residues located at the entrance and bottom of the cavity, respectively. This observation indicates that the lower region makes a rigid connection with PA whereas the entrance is more flexible. The result is in contrast to the binding of PA to intestinal fatty acid-binding protein, another member of the calycin superfamily, in which structural consolidation occurs upon ligand binding. On the other hand, the ability of beta LG to accommodate various hydrophobic ligands resembles that of GroEL, in which a large hydrophobic cavity and flexible binding site confer the ability to bind various hydrophobic substrates. Considering these observations, it is suggested that, in addition to the presence of the hydrophobic cavity, the plasticity of the entrance region makes possible the binding of hydrophobic ligands of various shapes. Thus, in contrast to the specific binding seen for many enzymes, beta LG provides an example of binding with low specificity but high affinity, which may play an important role in protein-ligand and protein-protein networks. (c) 2007 Elsevier Ltd. All rights reserved.
  • Flow-induced alignment of amyloid protofilaments revealed by linear dichroism, Rumi Adachi, Kei-ichi Yamaguchi, Hisashi Yagi, Kazumasa Sakurai, Hironobu Naiki, Yuji Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 282(12), 8978 - 8983, Mar. 2007 , Refereed
    Summary:uAmyloid fibrils underlying various serious amyloidoses including Alzheimer and prion diseases form characteristic deposits in which linear fibrils with an unbranched and rigid morphology associate laterally or radially, e.g. radial senile amyloid plaques of amyloid beta. To clarify the formation of these high order amyloid deposits, studying the rheology is important. A 22-residue K3 peptide fragment of beta(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, forms long and homogeneous protofilament-like fibrils in 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCI (pH similar to 2). Here, using circular dichroism and linear dichroism, we observed the flow-induced alignment of fibrils. Analysis of far- and near-UV linear dichroism spectra suggested that both the net pi-pi transition moment of the backbone carbonyl group and L(b) transition moment of the Tyr(26) side chain are oriented in parallel to the fibril axis, revealing the structural details of amyloid protofilaments. Moreover, the intensities of flow-induced circular dichroism or linear dichroism signals depended critically on the length and type of fibrils, suggesting that they are useful for detecting and characterizing amyloid fibrils.
  • Cores and pH-dependent dynamics of ferredoxin-NADP(+) reductase revealed by hydrogen/deuterium exchange, Young-Ho Lee, Kosuke Tamura, Masahiro Maeda, Masaru Hoshino, Kazumasa Sakurai, Satoshi Takahashi, Takahisa Ikegami, Toshiharu Hase, Yuji Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 282(8), 5959 - 5967, Feb. 2007 , Refereed
    Summary:NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD, values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD, values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD, 8.0, the daytime pH in chloroplasts, than at pD, 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.
  • Dynamics and mechanism of the ligand binding of bovine beta-lactoglobulin studied using heteronuclear NMR spectroscopy., J. Mol. Biol., J. Mol. Biol., 368 (1), 209-218, 2007
  • High-resolution crystal structure of beta2-microglobulin formed under physiological conditions., J. Biochem., J. Biochem., 142 (3), 413-419, 2007
  • Dynamics and mechanism of the Tanford transition of bovine beta-lactoglobulin studied using Heteronuclear NMR Spectroscopy, K Sakurai, Y Goto, JOURNAL OF MOLECULAR BIOLOGY, JOURNAL OF MOLECULAR BIOLOGY, 356(2), 483 - 496, Feb. 2006 , Refereed
    Summary:The Tanford transition is a conformational change of bovine beta-lactoglobulin (beta LG) occurring at around pH 7, identified originally on the basis of optical rotatory dispersion and the accessibility of a thiol group. X-ray analysis has suggested that a conformational change to the EF-loop is responsible for the Tanford transition, with the loop closing the hydrophobic cavity of the beta-barrel of the beta LG molecule below pH 7 and flipping to open the cavity above pH 7. To clarify the dynamics of this conformational change, NMR measurements were made at neutral pH. Since severe signal broadening due to monomer-dimer equilibrium prevented NMR measurements of wildtype beta LG at neutral pH, we searched for optimal sample conditions, finding that a disulfide bond-linked dimer of the mutant A34C gives an HSQC spectrum without signal broadening. The HSQC and CD spectra indicated that in overall conformation A34C is similar to wild-type beta LG, suggesting that the A34C dimer is a good model with which to study the structure and dynamics of the wild-type at neutral pH. The pH-dependent HSQC signal changes and Lipari-Szabo type relaxation analyses of the A34C dimer revealed that the conformational change to the EF-loop occurs above pH 7. We observed two types of motions in the EF-loop region; relatively fast (micro- to milliseconds) and slow (milliseconds or slower) conformational exchanges of the residues located in the hinge and top of the EF-loop regions, respectively. Furthermore, the GH-loop adjacent to the EF-loop exhibited conformational change at a pH slightly lower than that at which the EF-loop motions occurred. From these observations, we propose a three-step mechanism of conformational change in the EF-loop leading to the Tanford transition, in which the GH-loop conformational change, the cleavage of the hydrogen bonds at the hinge, and the flip of the EF-loop occur sequentially. (c) 2005 Elsevier Ltd. All rights reserved.
  • Conformational stability of amyloid fibrils of beta-microglobulin probed by guanidine-hydrochloride-induced unfolding, T Narimoto, K Sakurai, A Okamoto, E Chatani, M Hoshino, K Hasegawa, H Naiki, Y Goto, FEBS LETTERS, FEBS LETTERS, 576(3), 313 - 319, Oct. 2004 , Refereed
    Summary:Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. beta(2)-microglobulin (beta2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of beta2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Reversible unfolding of bovine beta-lactoglobulin mutants without a free thiol group, M Yagi, K Sakurai, C Kalidas, CA Batt, Y Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 278(47), 47009 - 47015, Nov. 2003 , Refereed
    Summary:Bovine beta-lactoglobulin (beta-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three beta-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type beta-lg. Urea-induced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 M urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type beta-lg in 8 M urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type beta-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of beta-lg more exactly under various conditions.
  • Manipulating monomer-dimer equilibrium of bovine β-lactoglobulin by amino acid substitution, Kazumasa Sakurai, Yuji Goto, Journal of Biological Chemistry, Journal of Biological Chemistry, 277(28), 25735 - 25740, Jul. 12 2002
    Summary:Bovine β-lactoglobulin, a major protein in cow's milk composed of nine β-strands (βA-βI) and one α-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular α-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation. Various I-strand mutants showed decreases in Kα, suggesting that the intermolecular α-sheet is essential for dimer formation. By substituting either Asp 33 or Arg40 on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in Ka was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg40 on one monomer and Asp33 on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg33 and ASp40 of different molecules. These results suggested that protein-protein interactions of bovine β-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.
  • Manipulating monomer-dimer equilibrium of bovine beta-lactoglobulin by amino acid substitution, K Sakurai, Y Goto, JOURNAL OF BIOLOGICAL CHEMISTRY, JOURNAL OF BIOLOGICAL CHEMISTRY, 277(28), 25735 - 25740, Jul. 2002 , Refereed
    Summary:Bovine beta-lactoglobulin, a major protein in cow's milk composed of nine beta-strands (betaA-betaI) and one a-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular beta-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation. Various I-strand mutants showed decreases in K-alpha, suggesting that the intermolecular beta-sheet is essential for dimer formation. By substituting either Asp(33) or Arg(40) on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in K-alpha was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg40 on one monomer and Asp(33) on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg(33) and Asp(40) of different molecules. These results suggested that protein-protein interactions of bovine beta-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding.
  • Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3, K Sakurai, M Oobatake, Y Goto, PROTEIN SCIENCE, PROTEIN SCIENCE, 10(11), 2325 - 2335, Nov. 2001
    Summary:Although bovine beta -lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO4 was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaCIO4, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta -lactoglobutin at pH 3.0 with NaClO4. The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degreesC and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaCIO4 the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of P-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.
  • Conformation and stability of thiol-modified bovine beta-lactoglobulin, K Sakai, K Sakurai, M Sakai, M Hoshino, Y Goto, PROTEIN SCIENCE, PROTEIN SCIENCE, 9(9), 1719 - 1729, Sep. 2000
    Summary:Bovine beta-lactoglobulin A assumes a dimeric native conformation at neutral pH, while the conformation at pH 2 is monomeric but still native. beta-Lactoglobulin A has a free thiol at Cysl21, which is buried between the beta-barrel and the C-terminal major alpha-helix. This thiol group was specifically reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of 1.0 M Gdn-HCl at pH 7.5, producing a modified beta-lactoglobulin (TNB-blg) containing a mixed disulfide bond with 5-thio-2-nitrobenzoic acid (TNB). The conformation and stability of TNB-blg were studied by circular dichroism (CD), tryptophan fluorescence, analytical ultracentrifugation, and one-dimensional H-1-NMR. The CD spectra of TNB-blg indicated disordering of the native secondary structure at pH 7.5, whereas a slight increase in the ct-helical content was observed at pH 2.0. The tryptophan fluorescence of TNB-blg was significantly quenched compared with that of the intact protein, probably by the energy transfer to TNB. Sedimentation equilibrium analysis indicated that, at neutral pH, TNB-blg is monomeric while the intact protein is dimeric. Tn contrast, at pH 2.0, both the intact beta-lactoglobulin and TNB-blg were monomeric. The unfolding transition of TNB-blg induced by Gdn-HCl was cooperative in both pH regions, although the degree of cooperativity was less than that of the intact protein. The H-1-NMR spectrum for TNB-blg at pH 3.0 was native-like, whereas the spectrum at pH 7.5 was similar to that of the unfolded proteins. These results suggest that modification of the buried thiol group destabilizes the rigid hydrophobic core and the dimer interface, producing a monomeric state that is native-like at pH 2.0 but is molten globule-like at pH 7.5. Upon reducing the mixed disulfide of TNB-blg with dithiothreitol, the intact beta-lactoglobulin was regenerated. TNB-blg will become a useful model to analyze the conformation and stability of the intermediate of protein folding.

Books etc

  • Molecular Structure of Amyloid Fibrils Revealed by Thermodynamics, Ikenoue, T, Lin, Y, Kinoshita, M, Sakurai, K, Goto, Y, Lee, Y.-H, Joint author, Chapter 5 in Protein-Protein Interactions (PPIs): Types, Methods for Detection and Analysis, Nova Science Publishers,   2016 , 9781634859707
  • アミロイド線維の構造と形成機構―研究の歴史と現在の試み, SAKURAI Kazumasa, GOTO Yuji, Joint author, 揺らぎ・ダイナミクスと生体機能 物理化学的視点から見た生体分子 11章, 化学同人,   2013 09
  • フォールディング入門-タンパク質の構造や機能の基盤, SAKURAI Kazumasa, GOTO Yuji, Joint author, タンパク質の一生集中マスター 第1章, 羊土社,   2007 03

Conference Activities & Talks

  • Heating-induced amyloid formation of b2-microglobulin at neutral pH, Noji, M. Sasahara, K. Yamaguchi, K. So, M. Sakurai, K. Kardos, J. Naiki, H. Goto, The 57th annual meeting of the Biophysical Society of Japan,   2019 09 25
  • Amyloid Fibrillation under complexity with coexisting b2-microglobulin and its proteolytic fragments, Muta, H. So, M. Sakurai, K. Kardos, J. Naiki, H. Goto, The 19th annual meeting of Protein Science Society Japan,   2019 06 25
  • Analysis of pH/pressure-dependent structural changes of a-synuclein based on chemical shift data, SAKURAI Kazumasa, The 19th annual meeting of Protein Science Society Japan,   2019 06 25
  • The mechanism of structural dynamics perturbations by the D76N mutation of β2-microglobulin revealed by pressure-NMR and MD simulations, SAKURAI Kazumasa, BK 21 PLUS-SKKU SYMPOSIUM“Cellular/Molecular Neuroscience and Human Disease”,   2019 04 29 , 招待有り
  • Analyses of pH/pressure-dependent structural changes of α-synuclein by chemical shift data, ABE Satoshi, SAKURAI Kazumasa, Ulm Meeting – Biophysics of Amyloid Formation,   2019 02 20
  • Insights into the hidden states of amyloidogenic proteins from pressure-induced changes, SAKURAI Kazumasa, Ulm Meeting – Biophysics of Amyloid Formation,   2019 02 20
  • Insights into the amyloidogenic intermediate states of β2-microglobulin and its pathogenic variant from pressure-induced folding/unfolding experiments, Ryosuke Tomiyama, Miwa Nakai, Akihiro Toyomasu, Kazumasa Sakurai, The 10th International Conference on High Pressure Biosicence and Biotechnology,   2018 09 18
  • Insights into “hidden” states of amyloidogenic proteins from the pressure-induced conformational changes, SAKURAI Kazumasa, The 4th International Symposium for Molecular Neurodegenerative Disease Research,   2018 08 22 , 招待有り
  • Comparison between conventional, quadratic analysis and chemical-shift-PCA on data of pressure-dependent chemical shift changes, SAKURAI Kazumasa, 8th International Meeting on Biomolecules under Pressure (IMBP),   2017 08 21
  • The establishment of a bacterial expression system of amyloid-dissociating p rotein, unfoldin, Sakurai, K, Nakata, S, Nakatani, A, Fujii, K, Hachiya, N,   2017 06 20
  • Amyloid Fibrillation in Promiscuous Systems Containing Various Amyloidogenic Peptides, SAKURAI Kazumasa, 第17回日本蛋白質科学会,   2017 06 20
  • Functional structure analysis of catalytic domain of endo1,3-β-glucanase, Miki, A, Inaba, S, Sakurai, K, Ishima, R, Oda, M, 日本農芸化学会2017年度大会,   2017 03 17
  • エンド‐1,3‐β‐グルカナーゼ触媒ドメインの機能構造解析, 三木彩子, 稲葉理美, 櫻井一正, 池上貴久, 伊島理枝子, 織田昌幸, 日本農芸化学会大会講演要旨集(Web),   2017 03 05
  • 複数のアミロイド性ペプチドの混在する複雑な系におけるアミロイド線維形成機構, Muta, H, So, M, Sakurai, K, Goto, Y, 第54回日本生物物理学会年会,   2016 11 26
  • Structural dynamics analysis of catalytic domain of endo-1,3-β-glucanase, Miki, A, Inaba, S, Sakurai, K, Oda, M, 第54回日本生物物理学会年会,   2016 11 26
  • Principal component analysis of chemical shift perturbation data of a pressure-dependent conformational change of amyloidogenic precursor state of β2-microglobulin, Sakurai, K, Maeno, H, Akasaka, K, XXVIIth International Conference on Magnetic Resonance in Biological Systems,   2016 08 21
  • Amyloid Fibrillation in Promiscuous Systems Containing Various Amyloidogenic Peptides, SAKURAI Kazumasa, XXVIIth International Conference on Magnetic Resonance in Biological Systems,   2016 08 21
  • 複数のアミロイド性ペプチドの混在する複雑な系におけるアミロイド線維形成機構, Muta, H, So, M, Sakurai, K, Goto, Y, 第16回日本蛋白質科学会,   2016 06 08
  • Insights into the pressure-induced conformational change of β2-microglobulin and its pathogenic variants, SAKURAI Kazumasa, Joint Symposium between the Institute of Protein Research and the Research School of Chemistry at the Australian National University,   2015 11 15 , 招待有り
  • 野生型及び病原性変異体β2ミクログロブリンの圧力変性反応の研究, 櫻井 一正, 豊増 明博, 前野 覚大, 橘 秀樹, 赤坂 一之, 第56回高圧討論会,   2015 11 10
  • Dissociation and Unfolding of Tobacco Mosaic Virus Coat Protein Assemblages, 深尾 博章, 櫻井 一正, 米澤 康滋, 藤澤 雅夫, 石橋 和大, 石川 雅之, 飯 哲夫, 橘 秀樹, 第53回日本生物物理学会年会,   2015 09 15
  • 野生型及び病原性変異体β2ミクログロブリンの圧力変性反応の研究, Sakurai, K, Toyomasu, A, Maeno, A, Tachibana, H, Akasaka, K, 第53回日本生物物理学会年会,   2015 09 14
  • 複数のアミロイド性ペプチドの混在する複雑な系におけるアミロイド線維形成機構, Muta, H, So, M, Sakurai, K, Goto, Y, 第53回日本生物物理学会年会,   2015 09 13
  • 蛋白質の機能構造として働く励起構造, Inaba, S, Maeno, A, Sakurai, K, Ikegami, T, Akasaka, K, Oda, M, 第53回日本生物物理学会年会,   2015 09 13
  • 過飽和を考慮した競争的な蛋白質凝集メカニズム, ADACHI NOBUYUKI, SO MASATOMO, SAKURAI KAZUMASA, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2015 05 26
  • 野生型及び病原性変異体β2ミクログロブリンの圧力変性反応の研究, SAKURAI KAZUMASA, TOYOMASU AKIHIRO, MAENO AKIHIRO, TACHIBANA HIDEKI, TACHIBANA HIDEKI, AKASAKA KAZUYUKI, 日本蛋白質科学会年会プログラム・要旨集,   2015 05 26
  • 1次元NMRによる深海菌ジヒドロ葉酸還元酵素の構造安定性の研究, SAKURAI KAZUMASA, OMAE EIJI, AKASAKA KAZUYUKI, 高圧討論会講演要旨集,   2014 11 10
  • 温度/圧力変化で見えるc‐Myb DNA結合ドメインの構造揺らぎの特性, INABA SATOMI, MAENO AKIHIRO, SAKURAI KAZUMASA, AKASAKA KAZUYUKI, ODA MASAYUKI, 高圧討論会講演要旨集,   2014 11 10
  • 温度変化実験により明らかとなったc‐Myb DNA結合ドメイン構造揺らぎの特性, INABA SATOMI, MAENO AKIHIRO, SAKURAI KAZUMASA, IKEGAMI TAKAHISA, FUKADA HARUMI, MORII HISAYUKI, AKASAKA KAZUYUKI, ODA MASAYUKI, 日本蛋白質科学会年会プログラム・要旨集,   2014 05 26
  • αシヌクレインが形成するアミロイド線維の温度に対する構造安定性の分子機構, IKENOUE TATSUYA, RI EIKO, KARDOS JOZSEF, SAKURAI KAZUMASA, YAGI HISASHI, KAWATA YASUSHI, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2013 05 31
  • 固体NMRによるβ2ミクログロブリンのアミロイド線維構造解析, SO MASATOMO, MATSUKI HIROMI, EGAWA FUMIKO, TODOKORO YASUTO, SAEKI MIWAKO, SAKURAI KAZUMASA, FUJIWARA TOSHIMICHI, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2013 05 31
  • βラクトグロブリンの折り畳みストラテジー, SAKURAI KAZUMASA, KITAYAMA HIROKI, YAGI MASANORI, IKEGAMI TAKAHISA, NISHIMURA CHIAKI, GOTO YUJI, Abstr Annu Meet NMR Soc Jpn,   2012 10 22
  • 細胞外シャペロンによるアミロイド線維形成抑制機構の解明, OZAWA DAISAKU, HASEGAWA KAZUHIRO, LEE YOUNG-HO, SAKURAI KAZUMASA, YANAGI KOTARO, OKOSHI TADAKAZU, GOTO YUJI, NAIKI HIRONOBU, 日本蛋白質科学会年会プログラム・要旨集,   2012 05 31
  • アミロイド線維形成時に現れる過渡的中間体のキャラクタリゼーション, YANAGI KOTARO, SAKURAI KAZUMASA, YOSHIMURA YUICHI, KONUMA TSUYOSHI, LEE YOUNG-HO, SUGASE KENJI, IKEGAMI TAKAHISA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2012 05 31
  • ウシβラクトグロブリンの非天然折り畳み中間体はスムーズな折り畳みに寄与する, SAKURAI KAZUMASA, FUJIOKA SHUNSUKE, KONUMA TSUYOSHI, YAGI MASANORI, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2012 05 31
  • アミロイド線維形成時に現れる過渡的中間体のキャラクタリゼーション, YANAGI KOTARO, SAKURAI KAZUMASA, YOSHIMURA YUICHI, KONUMA TSUYOSHI, LEE YOUNG-HO, SUGASE KENJI, IKEGAMI TAKAHISA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2012 05 31
  • チオフラビンTとレーザー光を利用したアミロイド線維の分解, OZAWA DAISAKU, KAJI YUICHI, YAGI HISASHI, SAKURAI KAZUMASA, KAWAKAMI TOORU, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2011 05 27
  • 溶液NMRによるβ2ミクログロブリンアミロイド線維形成のモノマー‐線維核相互作用機構の解明, SAKURAI KAZUMASA, YANAGI KOTARO, YOSHIMURA YUICHI, KONUMA TSUYOSHI, SUGASE KENJI, IKEGAMI TAKAHISA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2011 05 27
  • 伸長反応中間体の観察によるアミロイド構造伝播機構の解明, CHATANI ERI, KONUMA TSUYOSHI, ONISHI REINA, YAGI MASANORI, SAKURAI KAZUMASA, IKEGAMI TAKAHISA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2011 05 27
  • 超音波処理下で作製した微細アミロイド線維の構造および物性のキャラクタリゼーション, YOSHIMURA YUICHI, SAKURAI KAZUMASA, YAGI HISASHI, SO MASATOMO, LEE YOUNG-HO, OGI HIROTSUGU, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2011 05 27
  • 超音波によるアミロイド線維形成促進, SO MASATOMO, YAGI HISASHI, SAKURAI KAZUMASA, OGI HIROTSUGU, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2011 05 27
  • ヘキサフルオロイソプロパノールによるIAPPのアミロイド線維形成, YANAGI KOTARO, ASHIZAKI MIZUE, YAGI HISASHI, SAKURAI KAZUMASA, LEE YOUNG HO, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2011 05 27
  • 蛍光色素チオフラビンTとレーザー光によるアミロイド線維の分解, OZAWA DAISAKU, KAJI YUICHI, YAGI HISASHI, SAKURAI KAZUMASA, KAWAKAMI TOORU, NAIKI HIRONOBU, GOTO YUJI, 生化学,   2011
  • レーザー照射によるアミロイド線維の増殖と崩壊, YAGI HISASHI, OZAWA DAISAKU, SAKURAI KAZUMASA, KAJI YUICHI, KAWAKAMI TOORU, KUYAMA HIROKI, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2010 05 15
  • β2‐ミクログロブリンが形成するアミロイド線維の溶液NMRによる直接観察, YOSHIMURA YUICHI, SAKURAI KAZUMASA, RI EIKO, IKEGAMI TAKAHISA, CHATANI ERI, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2010 05 15
  • パルスラベルH/D交換法とNMRを用いたβ2ミクログロブリンの線維伸長中間体の解析, KONUMA TSUYOSHI, CHATANI ERI, YAGI MASANORI, SAKURAI KAZUMASA, IKEGAMI TAKAHISA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2010 05 15
  • 超音波による超高速アミロイド線維形成反応, SO MASATOMO, YAGI HISASHI, SAKURAI KAZUMASA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2010 05 15
  • ヒトIgG1 CH3ドメインのSS結合還元に伴う物性変化の研究, NAKAHATA RYOSUKE, SAKURAI KAZUMASA, RI EIKO, KARDOS JOZEEF, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2010 05 15
  • 超音波によるK3 peptideとα‐synucleinのアミロイド線維形成, MIZUNO AIKO, SO MASATOMO, YAGI HISASHI, SAKURAI KAZUMASA, KAWATA YASUSHI, NAIKI HIRONOBU, GOTO YUJI, 生化学,   2010
  • ヒトIgG1 CH3ドメインのSS結合の還元に伴う物性変化の研究, NAKAHATA RYOSUKE, SAKURAI KAZUMASA, LEE YOUNG-HO, MATSUMURA MASAZUMI, GOTO YUJI, 生化学,   2009 09 25
  • N‐terminal nucleophile(Ntn)‐hydrolases family(NylC)の耐熱化機構, SHIBATA HIROSHI, TANAKA YUSUKE, HASHIMOTO HARUKA, MATSUURA YUSUKE, SHIBATA NAOKI, LEE YOUNG-HO, SAKURAI KAZUMASA, KATO DAIICHIRO, TAKEO MASAHIRO, HIGUCHI YOSHIKI, GOTO YUJI, NEGORO SEIJI, 日本生物工学会大会講演要旨集,   2009 08 25
  • β2‐ミクログロブリンが形成するアミロイド線維の溶液NMRによる運動性の解析, YOSHIMURA YUICHI, SAKURAI KAZUMASA, CHATANI ERI, KAMEDA ATSUSHI, IKEGAMI TAKAHISA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2009 04 24
  • H/D交換法によるβ2ミクログロブリンのアミロイド線維形成中間体の検出, KONUMA TSUYOSHI, CHATANI ERI, YAGI MASANORI, ONISHI REINA, SAKURAI KAZUMASA, NAIKI HIRONOBU, GOTO YUJI, Abstr Annu Meet NMR Soc Jpn,   2008 11 12
  • β2ミクログロブリンのフラグメントが形成するアミロイド線維構造の構造解析, YOSHIMURA YUICHI, CHATANI ERI, SAKAI MIYO, SAKURAI KAZUMASA, KAMEDA ATSUSHI, YAMAGUCHI KEIICHI, UCHIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2008 05 23
  • β‐ラクトグロブリンのヘリックス中間体の寄与の解明, FUJIOKA SHUNSUKE, KONUMA TSUYOSHI, YAGI MASANORI, SAKURAI KAZUMASA, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2008 05 23
  • 重水素交換法を用いたアミロイド線維伸長反応機構の解析, YANAGI KOTARO, SAKURAI KAZUMASA, KAMEDA ATSUSHI, CHATANI ERI, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2008 05 23
  • Principal Component Analysis of the pH-Dependent Conformational Transitions of Bovine β-Lactoglobulin Monitored by Heteronuclear NMR, SAKURAI Kazumasa, KONUMA Tsuyoshi, GOTO Yuji,   2007 12
  • Relationship between pH-dependences of conformation and stability of bovine beta-lactoglobulin studied using fluorescence and NMR spectroscopies, SAKURAI Kazumasa, GOTO Yuji, Osaka University Macromolecular Symposium,   2007 07
  • アミロイド線維および形成過程の立体構造解析, CHATANI ERI, ONISHI REINA, KAMEDA ATSUSHI, YAGI HISASHI, BAN TADATO, SAKURAI KAZUMASA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2007 05 07
  • トリプトファン蛍光を用いたアミロイド線維伸長過程における中間体の解析, ONISHI REINA, CHATANI ERI, SAKURAI KAZUMASA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2007 05 07
  • ジスルフィド結合により二量体化されたウシβラクトグロブリンの遅いフォールディング反応, YAGI MASANORI, KAMEDA ATSUSHI, SAKURAI KAZUMASA, NISHIMURA CHIAKI, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2007 05 07
  • β2ミクログロブリンの部分ペプチドから成るf210アミロイド線維の重水素交換実験, MIEDA KOJI, KAMEDA ATSUSHI, YAMAGUCHI KEIICHI, SAKURAI KAZUMASA, NAIKI HIRONOBU, GOTO YUJI, 生化学,   2007
  • Promiscuous binding of ligands by beta-lactoglobulin involves hydrophobic interactions and plasticity, KONUMA Tsuyoshi, SAKURAI Kazumasa, GOTO Yuji, Fifth East Asia Biophysics Syposium & Forty-Fourth Annual Meeting of the Biophysical Society of Japan,   2006 11
  • Folding and unfolding kinetics of beta2-microglobulin as probed by tryptophan flourescence, SAKATA Michiko, CHATANI Eri, KAMEDA Atshushi, SAKURAI Kazumasa, NAIKI Hironobu, GOTO Yuji, Fifth East Asia Biophysics Syposium & Forty-Fourth Annual Meeting of the Biophysical Society of Japan,   2006 11
  • Relationship between pH-dependences of conformation and stability of bovine beta-lactoglobulin studied using fluorescence and NMR spectroscopies, SAKURAI Kazumasa, GOTO Yuji, Fifth East Asia Biophysics Syposium & Forty-Fourth Annual Meeting of the Biophysical Society of Japan,   2006 11
  • Orientation of amyloid fibrils formed by beta2-microglobulin and its peptide fragment in a flow, Adachi, R, Yamaguchi, K, Sakai, M, Yagi, H, Chatani, E, Sakurai, K, Naiki, H, Goto, Y, Fifth East Asia Biophysics Syposium & Forty-Fourth Annual Meeting of the Biophysical Society of Japan,   2006 11
  • 流れによるアミロイド線維の配向性と沈着, ADACHI RUMI, YAMAGUCHI KEIICHI, SAKURAI KAZUMASA, NAIKI HIRONOBU, GOTO YUJI, 日本蛋白質科学会年会プログラム・要旨集,   2006 03 31
  • NMR experiment of bovine β-Lactoglobulin under neutral pH, SAKURAI Kazumasa, GOTO Yuji, Osaka University Macromolecular Symposium on Nano-Structured Macromolecular Materials,   2004 07
  • Protein-Protein Interaction of beta-Lactoglobulin Studied by Amino Acid Substitution, SAKURAI Kazumasa, HOSHINO Masaru, GOTO Yuji, Osaka University Macromolecular Symposium on Nano-Structured Macromolecular Materials,   2001 11

Misc

  • Different aspects of protein molecules revealed by novel analysis of high-pressure NMR data, SAKURAI Kazumasa, The Review of High Pressure Science and Technology, 30, 12, 19,   2020 03 01 , Refereed, 招待有り
  • The pressure effect on the assemblies of protein molecules investigated by high-pressure NMR observation, Ryosuke Tomiyama, Kazumasa SAKURAI, Mem. Inst. Adv. Tech. Kindai Univ., 25, 1, 10,   2020 03 , Refereed
  • Identification of inter-residue interactions appeared in the early stage of folding process of b-lactoglobulin and its folding mutant, E44L, by using high-pressure NMR, SAKURAI Kazumasa, Mem. Inst. Adv. Tech. Kindai Univ., 24, 1, 10,   2019 03 , Refereed
  • Reanalysis of pressure-dependent chemical shift data of various proteins by PCA procedure, NAKANO Takayuki, SAKURAI Kazumasa, Mem. Inst. Adv. Tech. Kindai Univ., 23, 1, 10,   2018 03 , Refereed
  • エンド‐1,3‐β‐グルカナーゼ触媒ドメインの機能構造解析, 三木彩子, 稲葉理美, 櫻井一正, 池上貴久, 伊島理枝子, 織田昌幸, 日本農芸化学会大会講演要旨集(Web), 2017, ROMBUNNO.2J33p09 (WEB ONLY),   2017 03 05 , http://jglobal.jst.go.jp/public/201702229987679587
  • Comparison between conventional, quadratic analysis and chemical-shift-PCA on data of pressure-dependent chemical shift changes, SAKURAI Kazumasa, Mem. Inst. Adv. Tech. Kindai Univ., 22, 1, 12,   2017 03 , Refereed
  • Expression of proenzyme of microbial transglutaminase and its pressure-dependent unfolding behavior, Kazuma TAKASHIBA, SAKURAI Kazumasa, Mem. Inst. Adv. Tech. Kindai Univ., 21, 11, 22,   2016 05 , Refereed
  • Examination of the expression of novel chaperone protein Aip2p/Dld2p with methylotrophic yeast Pichia pastoris, SAKURAI Kazumasa, NAKATA Shoki, TOYOMASU Akihiro, TACHIBANA Hideki, Mem. Inst. Adv. Tech. Kindai Univ., 20, 31, 42,   2015 03 , Refereed
  • 温度変化実験により明らかとなったc‐Myb DNA結合ドメイン構造揺らぎの特性, 稲葉理美, 前野覚大, 櫻井一正, 池上貴久, 深田はるみ, 森井尚之, 赤坂一之, 織田昌幸, 日本蛋白質科学会年会プログラム・要旨集, 14th, 104,   2014 05 26 , http://jglobal.jst.go.jp/public/201402253136066851
  • High-pressure NMR measurements on human beta2-microglobulin, SAKURAI Kazumasa, MAENO Akihiro, AKASAKA Kazuyuki, Mem. Inst. Adv. Tech. Kindai Univ., 19, 35, 42,   2014 03 , Refereed
  • 3P061 The circumventing mechanism of the folding of β-lactoglobulin(01C. Protein: Property,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014)), Sakurai Kazumasa, Yagi Masanori, Nishimura Chiaki, Akasaka Kazuyuki, Goto Yuji, Seibutsu Butsuri, 54, 1,   2014 , http://ci.nii.ac.jp/naid/110009932930
  • 化学シフトデータの主成分解析による、蛋白質状態変化の洞察, SAKURAI Kazumasa, 日本核磁気共鳴学会学会誌, 4, 10, 19,   2013 , 招待有り
  • The Monomer-Seed Interaction Mechanism in the Formation of the .BETA.2-Microglobulin Amyloid Fibril Clarified by Solution NMR Techniques, YANAGI KOTARO, SAKURAI KAZUMASA, 生物物理, 53, 2, 107-108 (J-STAGE),   2013 , 10.2142/biophys.53.107, http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=201302249132879870
  • βラクトグロブリンの折り畳みストラテジー, 櫻井一正, 北山寛貴, 八木正典, 池上貴久, 西村千秋, 後藤祐児, Abstracts. Annual Meeting of the NMR Society of Japan, 51st, 114, 115,   2012 10 22 , https://jglobal.jst.go.jp/detail?JGLOBAL_ID=201302218399821266
  • アミロイド線維形成時に現れる過渡的中間体のキャラクタリゼーション, 柳浩太郎, 櫻井一正, 吉村優一, 小沼剛, LEE Young‐Ho, 菅瀬謙治, 池上貴久, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 12th, 12,   2012 05 31 , http://jglobal.jst.go.jp/public/201202227197330076
  • アミロイド線維形成時に現れる過渡的中間体のキャラクタリゼーション, 柳浩太郎, 櫻井一正, 吉村優一, 小沼剛, LEE Young‐Ho, 菅瀬謙治, 池上貴久, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 12th, 104,   2012 05 31 , http://jglobal.jst.go.jp/public/201202266679811808
  • A14 Ultrasonication-Dependent Acceleration of Amyloid Fibril Formation, So Masatomo, Yoshimura Yuichi, Yagi Hisashi, Sakurai Kazumasa, Goto Yuji, Ogi Hirotsugu, Naiki Hironobu, Proceedings of the Annual Meeting of the Japan Society of Sonochemistry, 21, 0, 85, 86,   2012 , http://ci.nii.ac.jp/naid/110009710750
    Summary:Amyloid fibrils are highly ordered assemblies of misfolded proteins associated with over 30 degenerative diseases including Alzheimer's diseases or type 2 diabetes. Fibrils form through nucleation and growth. This is similar to crystallization. Fibrillation takes a long time because of the high free-energy barrier to nucleation. Understanding the mechanisms of fibrillation is useful to treat amyloid associated diseases. However, studies have been limited by a long lag phase and low reproducibility of fibrillation. Ultrasonication can generate fibrils in a short time and a microplate reader can detect fibrils in many samples simultaneously. Here, we propose the amyloid-screening system with a combined use of ultrasonicator and microplate reader. Further, we show the correlation of fibrillation with ultrasonic amplitude. The results indicate that fibrillation is a physical reaction that is largely limited by high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will contribute to the understanding of the amyloidogenicity of various proteins and to the creation of therapeutic strategies against a wide range of degenerative diseases.
  • 超音波処理下で作製した微細アミロイド線維の構造および物性のキャラクタリゼーション, 吉村優一, 櫻井一正, 八木寿梓, 宗正智, LEE Young‐Ho, 荻博次, 内木宏信, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 11th, 101,   2011 05 27 , http://jglobal.jst.go.jp/public/201102248981942460
  • 伸長反応中間体の観察によるアミロイド構造伝播機構の解明, 茶谷絵理, 小沼剛, 大西玲奈, 八木正典, 櫻井一正, 池上貴久, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 11th, 34,   2011 05 27 , http://jglobal.jst.go.jp/public/201102255293571943
  • 溶液NMRによるβ2ミクログロブリンアミロイド線維形成のモノマー‐線維核相互作用機構の解明, 櫻井一正, 柳浩太郎, 吉村優一, 小沼剛, 菅瀬謙治, 池上貴久, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 11th, 104,   2011 05 27 , http://jglobal.jst.go.jp/public/201102265450218417
  • パルスラベルH/D交換法とNMRを用いたβ2ミクログロブリンの線維伸長中間体の解析, 小沼剛, 茶谷絵理, 八木正典, 櫻井一正, 池上貴久, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 10th, 171,   2010 05 15 , http://jglobal.jst.go.jp/public/201002265141575038
  • β2‐ミクログロブリンが形成するアミロイド線維の溶液NMRによる直接観察, 吉村優一, 櫻井一正, 李映昊, 池上貴久, 茶谷絵理, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 10th, 90,   2010 05 15 , http://jglobal.jst.go.jp/public/201002278518200470
  • 1P063 1YA0930 Identification of transient intermediates of the formation of the β2-microglobulin amyloid fibril by heteronuclear NMR techniques.(Protein:Property,Early Research in Biophysics Award Candidate Presentations,Early Research in Biophysics Award, SAKURAI Kazumasa, YANAGI Kotaro, YOSHIMURA Yuichi, KONUMA Tsuyoshi, IKEGAMI Takahisa, NAIKI Hironobu, GOTO Yuji, Seibutsu Butsuri, 50, 2,   2010 , http://ci.nii.ac.jp/naid/110008102299
  • β2‐ミクログロブリンが形成するアミロイド線維の溶液NMRによる運動性の解析, 吉村優一, 櫻井一正, 茶谷絵理, 亀田篤司, 池上貴久, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 9th, 74,   2009 04 24 , http://jglobal.jst.go.jp/public/200902230403026651
  • β2ミクログロブリンのフラグメントが形成するアミロイド線維構造の構造解析, 吉村優一, 茶谷絵理, 酒井美世, 櫻井一正, 亀田篤司, 山口圭一, 内木宏延, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 8th, 119,   2008 05 23 , http://jglobal.jst.go.jp/public/200902270959320712
  • Rise of amyloid structural biology, YAGI HISASHI, SAKURAI KAZUMASA, GOTO YUJI, 蛋白質 核酸 酵素, 52(12):1445-1453, 12, 1445, 1453,   2007 10 , 招待有り, http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=200902298138868207
  • ジスルフィド結合により二量体化されたウシβラクトグロブリンの遅いフォールディング反応, 八木正典, 亀田篤司, 櫻井一正, 西村千秋, 後藤祐児, 日本蛋白質科学会年会プログラム・要旨集, 7th, 102,   2007 05 07 , https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200902246105992093
  • 1P095 Analyses of the folding reaction of the bovine β-lactoglobulin mutant using pulse-labeling H/D exchange, Yagi M, Kameda A, Sakurai K, Nishimura C, Goto Y, Seibutsu Butsuri, 45, 0,   2005 , http://ci.nii.ac.jp/naid/110004571076
  • 実はよくわかっていない折り畳みの”スタート”, SAKURAI Kazumasa, 生物工学会誌, 6, 307,   2005 , 招待有り
  • in vitro フォールディング, SAKURAI Kazumasa, GOTO Yuji, 細胞工学, 23, 1364, 1369,   2004 , 招待有り
  • Structural dynamics and folding of beta-lactoglobulin probed by heteronuclear NMR, Kazumasa Sakurai, Tsuyoshi Konuma, Masanori Yagi, Yuji Goto, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1790, 6, 527, 537,   2009 06 , 10.1016/j.bbagen.2009.04.003
    Summary:Bovine P-lactoglobulin (beta LG) has been one of the most extensively studied proteins in the history of protein science mainly because its abundance in cow's milk makes it readily available to researchers. However, compared to other textbook proteins, progress in the study of beta LG has been slow because of obstacles such as a low reversibility from denaturation linked with thiol-disulfide exchange or monomer-dimer equilibrium preventing a detailed NMR analysis. Recently, the expression of various types of recombinant beta LGs combined with heteronuclear NMR analysis has significantly improved understanding of the physico-chemical properties of beta LG. In this review, we address several topics including pH-dependent structural dynamics, ligand binding, and the complex folding mechanism with non-native intermediates. These unique properties might be brought about by conformational frustration of the beta LG structure, partly attributed to the relatively large molecular size of beta LG. We expect studies with beta LG to continue to reveal various important findings, difficult to obtain with small globular proteins, leading to a more comprehensive understanding of the conformation, dynamics and folding of proteins. (C) 2009 Elsevier B.V. All rights reserved.

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(若手研究(B)), Characterization of the transient intermediate of the β2-microglobulin amyloid fibril using solution NMR techniques, The purpose of this study is to clarify how β2-Microglobulin (β2m), associated with the dialysis-related amyloidosis, changes its conformation when the disease develops by using nuclear magnetic resonance. It has been believed that β2m forms amyloid fibril through a partially structured state. However, from the present data, we suggested that the partially structured state should be "activated" to be extensively unfolded state, in which the hydrophobic residues are exposed to associate with the seed.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(若手研究(B)), Structural analysis of growing-end intermediate of amyloid fibril using NMR
  • Basic Science Research Program, Study on high-order structure formation of protein