Researchers Database

shibayama naoya

    PhysiologyMolecularPhysiology&Biophysics Professor
Last Updated :2021/12/07

Researcher Information

Degree

  • (BLANK)

URL

Research funding number

  • 20196439

J-Global ID

Research Interests

  • ゾル・ゲル法   EA4   生物時計   ヘモグロビン   アロステリー   Sol-gel   EA4   Biological clock   Hemoglobin   Allostery   

Research Areas

  • Life sciences / Biophysics
  • Life sciences / Structural biochemistry

Academic & Professional Experience

  • Jichi Medical UniversityLecturer

Education

  •        - 1985  Osaka University  基礎工学研究科  物理系専攻 生物工学分野
  •        - 1985  Osaka University  Graduate School, Division of Engineering Science
  •        - 1983  Osaka University  School of Engineering Science Direct Affiliates  生物工学科
  •        - 1983  Osaka University  Faculty of Engineering Science

Association Memberships

  • American Society of Biochemistry and Molecular Biology   日本生化学会   日本生物物理学会   

Published Papers

  • Tadayoshi Karasawa, Akira Kawashima, Fumitake Usui-Kawanishi, Sachiko Watanabe, Hiroaki Kimura, Ryo Kamata, Koumei Shirasuna, Yutaro Koyama, Ayana Sato-Tomita, Takashi Matsuzaka, Hiroshi Tomoda, Sam-Yong Park, Naoya Shibayama, Hitoshi Shimano, Tadashi Kasahara, Masafumi Takahashi
    Arteriosclerosis, Thrombosis, and Vascular Biology 38 (4) 744 - 756 1524-4636 2018/04 [Refereed][Not invited]
     
    Objective - Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1β (interleukin 1β) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. Approach and Results - The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1β release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β-deficient mice. Conclusions - These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.
  • Naoya Shibayama, Mio Ohki, Jeremy R. H. Tame, Sam-Yong Park
    JOURNAL OF BIOLOGICAL CHEMISTRY 292 (44) 18258 - 18269 0021-9258 2017/11 [Refereed][Not invited]
     
    Although X-ray crystallography is the most commonly used technique for studying the molecular structure of proteins, it is not generally able to monitor the dynamic changes or global domain motions that often underlie allostery. These motions often prevent crystal growth or reduce crystal order. We have recently discovered a crystal form of human hemoglobin that contains three protein molecules allowed to express a full range of quaternary structures, whereas maintaining strong X-ray diffraction. Here we use this crystal form to investigate the effects of two allosteric effectors, phosphate and bezafibrate, by tracking the structures and functions of the three hemoglobin molecules following the addition of each effector. The X-ray analysis shows that the addition of either phosphate or bezafibrate not only induces conformational changes in a direction from a relaxed-state to a tense-state, but also within relaxed-state populations. The microspectrophotometric O-2 equilibrium measurements on the crystals demonstrate that the binding of each effector energetically stabilizes the lowest affinity conformer more strongly than the intermediate affinity one, thereby reducing the O-2 affinity of tense-state populations, and that the addition of bezafibrate causes an approximate to 5-fold decrease in the O-2 affinity of relaxed-state populations. These results show that the allosteric pathway of hemoglobin involves shifts of populations rather than a unidirectional conversion of one quaternary structure to another, and that minor conformers of hemoglobin may have a disproportionate effect on the overall O-2 affinity.
  • Mio Ohki, Ayana Sato-Tomita, Shigeru Matsunaga, Mineo Iseki, Jeremy R. H. Tame, Naoya Shibayama, Sam-Yong Park
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 114 (32) 8562 - 8567 0027-8424 2017/08 [Refereed][Not invited]
     
    The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light-and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.
  • Satoru Fujiwara, Toshiyuki Chatake, Tatsuhito Matsuo, Fumiaki Kono, Taiki Tominaga, Kaoru Shibata, Ayana Sato-Tomita, Naoya Shibayama
    JOURNAL OF PHYSICAL CHEMISTRY B 121 (34) 8069 - 8077 1520-6106 2017/08 [Refereed][Not invited]
     
    Hemoglobin, the vital O-2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency in the former than in the latter. Our results reveal the presence of rapid large-scale motions in hemoglobin and further demonstrate that this internal mobility is governed allosterically by the ligation state of the heme group.
  • Mio Ohki, Kanako Sugiyama, Fumihiro Kawai, Hitomi Tanaka, Yuuki Nihei, Satoru Unzai, Masumi Takebe, Shigeru Matsunaga, Shin-ichi Adachi, Naoya Shibayama, Zhiwen Zhou, Ryuta Koyama, Yuji Ikegaya, Tetsuo Takahashi, Jeremy R. H. Tame, Mineo Iseki, Sam-Yong Park
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 113 (24) 6659 - 6664 0027-8424 2016/06 [Refereed][Not invited]
     
    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 angstrom across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.
  • Ayana Sato-Tomita, Naoya Shibayama, Naohisa Happo, Koji Kimura, Takahiro Okabe, Tomohiro Matsushita, Sam-Yong Park, Yuji C. Sasaki, Kouichi Hayashi
    REVIEW OF SCIENTIFIC INSTRUMENTS 87 (6) 063707  0034-6748 2016/06 [Refereed][Not invited]
     
    Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metal-loprotein samples are described. Human hemoglobin, an alpha(2)beta(2) tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A chi-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 x 6 x 3 mm(3)) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0 degrees-70 degrees. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules. Published by AIP Publishing.
  • Takahiro Okabe, Seiichi Tsukamoto, Kazuo Fujiwara, Naoya Shibayama, Masamichi Ikeguchi
    BIOCHEMISTRY 53 (23) 3858 - 3866 0006-2960 2014/06 [Refereed][Not invited]
     
    Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine beta-lactoglobulin, human tear lipocalin, bovine alpha-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.
  • Naoya Shibayama, Kanako Sugiyama, Jeremy R. H. Tame, Sam-Yong Park
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 136 (13) 5097 - 5105 0002-7863 2014/04 [Refereed][Not invited]
     
    Allostery in many oligomeric proteins has been postulated to occur via a ligand-binding-driven conformational transition from the tense (T) to relaxed (R) state, largely on the basis of the knowledge of the structure and function of 2 1 hemoglobin, the most thoroughly studied of all allosteric proteins. However, a growing body of evidence suggests that hemoglobin possesses a variety of intermediates between the two end states. As such intermediate forms coexist with the end states in dynamic equilibrium and cannot be individually characterized by conventional techniques, very little is known about their properties and functions. Here we present complete structural and functional snapshots of nine equilibrium conformers of human hemoglobin in the halfliganded and fully liganded states by using a novel combination of X-ray diffraction analysis and microspectrophotometric 02 equilibrium measurements on three isomorphous crystals, each capturing three distinct equilibrium conformers. Notably, the conformational set of this crystal form varies according to shifts in the allosteric equilibrium, reflecting the differences in hemoglobin ligation state and crystallization solution conditions. We find that nine snapshot structures cover the complete conformational space of hemoglobin, ranging from T to R2 (the second relaxed quaternary structure) through R, with various relaxed intermediate forms between R and R2. Moreover, we find a previously unidentified intermediate conformer, between T and R, with an intermediate O2 affinity, sought by many research groups over a period of decades. These findings reveal a comprehensive picture of the equilibrium conformers and transition pathway for human hemoglobin.
  • Kenta Yamada, Haruto Ishikawa, Misao Mizuno, Naoya Shibayama, Yasuhisa Mizutani
    JOURNAL OF PHYSICAL CHEMISTRY B 117 (41) 12461 - 12468 1520-6106 2013/10 [Refereed][Not invited]
     
    Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the beta subunits. Changes in the resonance Raman spectra of the alpha heme and the beta Ni-heme groups in the hybrid hemoglobin were observed upon CO photolysis in the alpha subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the upsilon(Fe-His) and the gamma(7) band of alpha heme was similar to that of unsubstituted hemoglobin, suggesting that substitution with Ni-heme did not perturb the allosteric dynamics of the hemoglobin. In the beta subunits, no structural change in the Ni-heme was observed until 1 mu s. In the microsecond regime, temporal evolution of the frequencies of the upsilon(Ni-His) and the gamma(7) band of beta Ni heme was observed concomitant with an R T quaternary change at about 20 The changes in the upsilon(Fe-His) and upsilon(Ni-His) frequencies of the a and beta subunits with the common time constant of similar to 20 mu s indicated that the proximal tension imposed on the bond between the heme and the proximal histidine strengthened upon the quaternary changes in both the alpha and the beta subunits concertedly. This observation is consistent with the Perutz mechanism for allosteric control of oxygen binding in hemoglobin and, thus, is the first real-time observation of the mechanism. Protein dynamics and allostery based on the observed time-resolved spectra also are discussed.
  • Jiro Kikuchi, Naoya Shibayama, Satoshi Yamada, Taeko Wada, Masaharu Nobuyoshi, Tohru Izumi, Miyuki Akutsu, Yasuhiko Kano, Kanako Sugiyama, Mio Ohki, Sam-Yong Park, Yusuke Furukawa
    PLOS ONE 8 (4) e60649  1932-6203 2013/04 [Refereed][Not invited]
     
    The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (beta 1, beta 2 and beta 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the beta 5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a beta 5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.
  • Naoya Shibayama
    FEBS LETTERS 586 (1) 74 - 78 0014-5793 2012/01 [Refereed][Not invited]
     
    To investigate the conformational changes in human tetrameric (alpha beta)(2) hemoglobin upon binding of the first two ligands, we have measured the kinetics of reactions between 4,4'-dithiodipyridine and beta 93Cys sulfhydryl groups of four diliganded hemoglobins by using CO-bound Fe(II)-Ni(II) hybrids with and without beta-beta cross-linking. The data show that all the diliganded intermediates have high sulfhydryl reactivities, which are greater than or equal to that for the fully-liganded end state, especially when containing liganded alpha subunit(s). The results also reveal that both the asymmetrically (alpha 1 beta 1 and alpha 1 beta 2) diliganded species show similar high rates of sulfhydryl reactivity and biphasic kinetics, suggesting a new conformation but only slight functional distortion caused by asymmetric ligation. (C) 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Naoya Shibayama, Kanako Sugiyama, Sam-Yong Park
    JOURNAL OF BIOLOGICAL CHEMISTRY 38 286 (38) 33661 - 33668 0021-9258 2011/09 [Refereed][Not invited]
     
    Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (beta 6 Glu -> Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 angstrom resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K-4) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2-3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.
  • 柴山修哉
    人工血液 18 (3) 102 - 109 1341-1594 2011/01 [Refereed][Not invited]
  • Andrey Kovalevsky, Toshiyuki Chatake, Naoya Shibayama, Sam-Yong Park, Takuya Ishikawa, Marat Mustyakimov, S. Zoe Fisher, Paul Langan, Yukio Morimoto
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 66 1144 - 1152 0907-4449 2010/11 [Refereed][Not invited]
     
    The protonation states of the histidine residues key to the function of deoxy (T-state) human hemoglobin have been investigated using neutron protein crystallography. These residues can reversibly bind protons, thereby regulating the oxygen affinity of hemoglobin. By examining the OMIT F (o) - F (c) and 2F (o) - F (c) neutron scattering maps, the protonation states of 35 of the 38 His residues were directly determined. The remaining three residues were found to be disordered. Surprisingly, seven pairs of His residues from equivalent alpha or beta chains, alpha His20, alpha His50, alpha His58, alpha His89, beta His63, beta His143 and beta His146, have different protonation states. The protonation of distal His residues in the alpha(1)beta(1) heterodimer and the protonation of alpha His103 in both subunits demonstrates that these residues may participate in buffering hydrogen ions and may influence the oxygen binding. The observed protonation states of His residues are compared with their delta pK (a) between the deoxy and oxy states. Examination of inter-subunit interfaces provided evidence for interactions that are essential for the stability of the deoxy tertiary structure.
  • Andrey Y. Kovalevsky, Toshiyuki Chatake, Naoya Shibayama, Sam-Yong Park, Takuya Ishikawa, Marat Mustyakimov, Zoe Fisher, Paul Langan, Yukio Morimoto
    JOURNAL OF MOLECULAR BIOLOGY 398 (2) 276 - 291 0022-2836 2010/04 [Refereed][Not invited]
     
    We have investigated the protonation states of histidine residues (potential Bohr groups) in the deoxy form (T state) of human hemoglobin by direct determination of hydrogen (deuterium) positions with the neutron protein crystallography technique. The reversible binding of protons is key to the allosteric regulation of human hemoglobin. The protonation states of 35 of the 38 His residues were directly determined from neutron scattering omit maps, with 3 of the remaining residues being disordered. Protonation states of 5 equivalent His residues-alpha His20, alpha His50, alpha His89, beta His143, and beta His146-differ between the symmetry-related globin subunits. The distal His residues, alpha His58 and beta His63, are protonated in the alpha 1 beta 1 heterodimer and are neutral in alpha 2 beta 2. Buried residue alpha His103 is found to be protonated in both subunits. These distal and buried residues have the potential to act as Bohr groups. The observed protonation states of His residues are compared to changes in their pK(a) values during the transition from the T to the R state and the results provide some new insights into our understanding of the molecular mechanism of the Bohr effect. Published by Elsevier Ltd.
  • Hideharu Akazaki, Yoshio Futami, Naoya Shibayama, Ikuko Shirasaki, Harumi Nakade, Hirotaka Chida, Wataru Hakamata, Sam-Yong Park, Toshiyuki Nishio, Tadatake Oku
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 72 (10) 2791 - 2794 0916-8451 2008/10 [Refereed][Not invited]
     
    To characterize a diheme cytochrome c(4) of unknown functional of the Vibrio genus for the first time, the Vibrio parahaemolyticus cytochrome c(4) was overexpressed in Escherichia coli periplasm using the endogenous signal sequence. The physicochemical properties of the purified recombinant protein, viz., molecular mass.. UV/Vis, and CD spectra, and the redox potentials of the N- and C-terminal domain hemes were determined.
  • Eiji Obayashi, Hisashi Yoshida, Fumihiro Kawai, Naoya Shibayama, Atsushi Kawaguchi, Kyosuke Nagata, Jeremy R. H. Tame, Sam-Yong Park
    NATURE 454 (7208) 1127 - U57 0028-0836 2008/08 [Refereed][Not invited]
     
    Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments(1). Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication(2-4). PB1 carries the polymerase active site, PB2 includes the capped- RNA recognition domain, and PA is involved in assembly of the functional complex(5-7), but so far very little structural information has been reported for any of them(8-11). We describe the crystal structure of a large fragment of one subunit ( PA) of influenza A RNA polymerase bound to a fragment of another subunit ( PB1). The carboxy- terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino- terminal residues of PB1 can fit by forming a 3(10) helix.
  • Naoya Shibayama
    FEBS LETTERS 582 (17) 2668 - 2672 0014-5793 2008/07 [Refereed][Not invited]
     
    beta-Lactoglobulin is a predominantly beta-sheet protein that folds by forming excess alpha-helices within milliseconds. In this study, the refolding of beta-lactoglobulin was dramatically decelerated by entrapping in wet nanoporous silica gel matrices, and monitored on a time scale of minutes or hours by far-UV circular dichroism spectroscopy. Analysis of kinetics and transient spectra allowed to define the sequence of folding events that consist of alpha-helical formation, beta-sheet core formation, and alpha-to-beta transition. The results suggest that the initially formed alpha-helices, presumably including the native alpha-helix, help to guide the formation of the adjacent beta-sheet core. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Naoya Shibayama
    BIOCHEMISTRY 47 (21) 5784 - 5794 0006-2960 2008/05 [Refereed][Not invited]
     
    Resolving the complete folding pathway of a protein is a major challenge to conventional experimental methods because of the rapidity and complexity of folding. Here, we show that entrapment of the protein cytochrome c in wet, optically transparent, porous silica gel matrices has enabled a dramatic expansion, to days or weeks, of the folding time, allowing direct observation of the entire folding pathway using a combination of three spectroscopic techniques, far-ultraviolet circular dichroism, tryptophan fluorescence, and Soret absorption spectroscopy. During refolding in silica gels, collapse and helix formation occur in a stepwise manner, as observed in aqueous solution. Analysis of kinetics and transient spectra indeed reveals a sequence of four distinct intermediates with progressively increasing degrees of folding, two of which closely resemble those previously characterized in solution, namely, the early collapsed and the molten globule intermediates. The other two are the precollapsed and pre-molten globule intermediates that may escape detection by conventional kinetic methods. Interestingly, varying the strength of the gel network has a dramatic effect on the folding time, but no significant effect on the structural features of each folding intermediate, indicating that the interaction between the protein and gel matrix has no measurable effect on the folding pathway. These results better define the major pathway of cytochrome c folding. In addition, in this paper we present the results of the application of this method to a simple, apparent two-state folder ubiquitin, helping to interpret the results for cytochrome c.
  • Toshiki Hiraki, Naoya Shibayama, Satoko Akashi, Sam-Yong Park
    JOURNAL OF MOLECULAR BIOLOGY 377 (3) 630 - 635 0022-2836 2008/03 [Refereed][Not invited]
     
    Many insects pass the winter in an arrested developmental stage called diapause, either as eggs, as pupae, or even as adults. Exposure to the prolonged cold of winter is required to permit awakening from diapause in the spring. In the diapause eggs of the silkworm Bombyx mori, a metalloglycoprotein, esterase A4 (EA4), has been suggested to serve as a cold-duration clock because its characteristic ATPase activity is transiently elevated at the end of the necessary cold period. This timer property of EA4 is known to start with the dissociation of an inhibitory peptide (called "peptidyl inhibitory needle") under cold conditions, but its time-measuring mechanism is completely unknown. Here we present the crystal structures and functional properties of EA4 with and without glycosylation. We show that EA4 is a homodimeric ATPase, with each subunit consisting of a copper-zinc superoxide dismutase fold. There is an additional short N-terminal region that is capable of binding one more copper ion, suggesting a timer mechanism in which this ion is involved. The sugar chain appears to reinforce the binding of peptidyl inhibitory needle, which may in turn stabilize the initial conformation of the N-terminal domain, explaining the requirement for glycosylation and for the peptide to set the clock. (C) 2008 Elsevier Ltd. All rights reserved.
  • Toshiyuki Chatake, Naoya Shibayama, Sam-Yong Park, Kazuo Kurihara, Taro Tamada, Ichiro Tanaka, Nobuo Niimura, Ryota Kuroki, Yukio Morimoto
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 129 (48) 14840 - + 0002-7863 2007/12 [Refereed][Not invited]
     
    The protonation states of buried histidine residues in human deoxyhemoglobin were unambiguously identified by using a neutron crystallographic technique. Unexpectedly, the neutron structure reveals that both the alpha- and beta-distal histidines (His alpha 58 and His beta 63) adopt a positively charged, fully (doubly) protonated form, suggesting their contribution to the Bohr effect. In addition, the neutron data provide an accurate picture of the alpha(1)beta(1) hydrogen-bonding network and allow us to observe unambiguously the nature of the intradimeric interactions at an atomic level.
  • Toshiki Hiraki, Naoya Shibayama, Young-Ho Yoon, Kyung-Mook Yun, Toshiro Hamamoto, Jeremy R. H. Tame, Sam-Yong Park
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 63 734 - 736 1744-3091 2007/09 [Refereed][Not invited]
     
    Esterase A4 ( EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 angstrom resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2(1), with unit- cell parameters a = 47.1, b = 73.9, c = 47.4 angstrom, beta = 104.1 degrees. With one dimer per asymmetric unit, the crystal volume per unit protein weight ( V-M) is 2.3 angstrom(3) Da(-1) and the solvent content is 47%.
  • Sam-Yong Park, Takeshi Yokoyama, Naoya Shibayama, Yoshitsugu Shiro, Jeremy R. H. Tame
    JOURNAL OF MOLECULAR BIOLOGY 360 (3) 690 - 701 0022-2836 2006/07 [Refereed][Not invited]
     
    The most recent refinement of the crystallographic structure of oxyhaemoglobin (oxyHb) was completed in 1983, and differences between this real-space refined model and later R state models have been interpreted as evidence of crystallisation artefacts, or numerous sub-states. We have refined models of deoxy, oxy and carbonmonoxy Hb to 1.25 angstrom resolution each, and compare them with other Hb structures. It is shown that the older structures reflect the software used in refinement, and many differences with newer structures are unlikely to be physiologically relevant. The improved accuracy of our models clarifies the disagreement between NMR and X-ray studies of oxyHb, the NMR experiments suggesting a hydrogen bond to exist between the distal histidine and oxygen ligand of both the alpha and beta-subunits. The high-resolution crystal structure also reveals a hydrogen bond in both subunit types, but with subtly different geometry which may explain the very different behaviour when this residue is mutated to glycine in a. or globin. We also propose a new set of relatively fixed residues to act as a frame of reference; this set contains a similar number of atoms to the well-known "BGH" frame yet shows a much smaller rmsd value between R and T state models of HbA. (c) 2006 Elsevier Ltd. All rights reserved.
  • SY Park, N Shibayama, T Hiraki, JRH Tame
    BIOCHEMISTRY 43 (27) 8711 - 8717 0006-2960 2004/07 [Refereed][Not invited]
     
    A number of ligand binding studies of human adult hemoglobin (HbA) cross-linked between Lys 82beta(1) and Lys 82beta(2) with bis(3,5-dibromosalicyl)fumarate have been reported. The oxygen binding properties of native HbA, including the cooperativity and Bohr effect, are not substantially changed by the modification, provided care is taken to remove electrophoretically silent impurities arising from side reactions. We have refined the high-resolution structure of this modified Hb and found it adopts the T state when crystallized in the absence of heme ligands, contrary to a previously published structure. These results suggest the slightly altered crystal form determined previously may be due to unremoved side products of the cross-linking reaction with high oxygen affinity. Two nickel-substituted Hbs cross-linked in the same way have also been crystallized in the presence of carbon monoxide, which binds only to the ferrous heme. In the case of the nickel-substituted alpha subunit, the absence of a covalent link between the central metal of the heme and the proximal histidine leads to a new conformation of the histidine stabilized by a water molecule. This structure may mimic that of partially NO-liganded species of HbA; however, overall, the changes are highly localized, and both doubly ligated species are in the T conformation.
  • SHIBAYAMA Naoya
    Biophysics 44 (3) 108 - 112 0582-4052 2004/05 [Not refereed][Not invited]
     
    The binding of ligands by proteins is accompanied by rapid structural changes that are essential to function. A recent crystallographic study has revealed the ligation-linked protein motions in an allosteric protein, human hemoglobin, in both allosteric forms (T and R) upon photolysis of bound CO at cryogenic temperatures. The results show how differently the α and β subunits, within each allosteric form, respond to loss of ligand, and where the free ligand lies, establishing that the mechanism of protein control of ligand binding is radically different between the subunits.
  • S Saigo, N Shibayama
    BIOCHEMISTRY 42 (32) 9669 - 9676 0006-2960 2003/08 [Refereed][Not invited]
     
    Theory and simulations predict that the folding kinetics of protein-like heteropolymers become nonexponential and glassy (i.e., controlled by escape from different low-energy misfolded states) at low temperatures, but there was little experimental evidence for such behavior of proteins. We have developed a stopped-flow instrument working reliably down to -40 degreesC with high mixing capability and applied it to study the refolding kinetics of horse cytochrome c (cyt c) and hen egg white lysozyme at temperatures below 0 degreesC in the presence of antifreeze NaCl, LiCl, or ethylene glycol and above 0 degreesC in the presence and absence of antifreeze. The refolding was initiated by rapid dilution of the guanidine hydrochloride unfolded proteins, and the kinetics were monitored by intrinsic tryptophan fluorescence. Highly nonexponential kinetics extended over 3 decades in time (0.01-10 s) were observed in the early phases of the refolding of cyt c and lysozyme in the temperature range of -35 to 5 degreesC. These results are in agreement with the theoretical prediction, suggesting that the folding energy landscapes of these proteins are rugged in the upper portions.
  • S Adachi, SY Park, JRH Tame, Y Shiro, N Shibayama
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 (12) 7039 - 7044 0027-8424 2003/06 [Refereed][Not invited]
     
    Human Hb, an alpha(2)beta(2) tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery, in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the a subunit than in the beta subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state beta heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.
  • N Shibayama, S Saigo
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 125 (13) 3780 - 3783 0002-7863 2003/04 [Refereed][Not invited]
     
    A comparison of the O-2 equilibrium curves of sperm-whale myoglobin locked in the liganded (CO-bound) and unliganded (deoxy) conformations by encapsulation in a wet porous sol-gel silica reveals a marked difference between them. The CO-bound state locked myoglobin showed a nearly monophasic (hyperbolic) O-2 equilibrium curve with a dissociation constant of 0.2 Torr, which is smaller than that of myoglobin in solution (0.5 Torr). On the other hand, the deoxy state-locked myoglobin exhibited a multiphasic O-2 equilibrium curve that can be represented by a sum of three independent components with dissociation constants of 0.19, 0.90, and 44 Torr, respectively, indicating that deoxymyoglobin exists in multiple conformations. These results show that myoglobin can be frozen into ligand-dependent conformational populations at room temperature in the wet sol-gel and suggest that the overall O-2 equilibrium properties of myoglobin in solution are generated by a redistribution of protein conformational populations in response to ligand binding.
  • N Shibayama, S Miura, JRH Tame, T Yonetani, SY Park
    JOURNAL OF BIOLOGICAL CHEMISTRY 277 (41) 38791 - 38796 0021-9258 2002/10 [Refereed][Not invited]
     
    Bezafibrate, an antilipidemic drug, is known as a potent allosteric effector of hemoglobin. The previously proposed mechanism for the allosteric potency of this drug was that it stabilizes and constrains the T-state of hemoglobin by specifically binding to the large central cavity of the T-state. Here we report a new allosteric binding site of fully liganded R-state hemoglobin for this drug. The high resolution crystal structure of horse carbonmonoxyhemoglobin in complex with bezafibrate reveals that the bezafibrate molecule lies near the surface of the E-helix of each a subunit and the complex maintains the quaternary structure of the R-state. Binding is caused by the close fit of bezafibrate into the binding pocket, which is composed of some hydrophobic residues and the heme edge, suggesting the importance of hydrophobic interactions. Upon binding of bezafibrate, the distance between Fe and the Ne, of distal His E7(alpha58) is shortened by 0.22 Angstrom in the a subunit, whereas no significant structural changes are transmitted to the 13 subunit. Oxygen equilibrium studies of R-state-locked hemoglobin with bezafibrate in a wet porous sol-gel indicate that bezafibrate selectively lowers the oxygen affinity of one type of subunit within the R-state, consistent with the structural data. These results disclose a new allosteric mechanism of bezafibrate and offer the first demonstration of how the allosteric effector interacts with R-state hemoglobin.
  • S Nagatomo, M Nagai, N Shibayama, T Kitagawa
    BIOCHEMISTRY 41 (31) 10010 - 10020 0006-2960 2002/08 [Refereed][Not invited]
     
    The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the (x heme, while it was similarly induced by binding of CO and NO to the,8 heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place In a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.
  • KM Yun, H Morimoto, N Shibayama
    JOURNAL OF BIOLOGICAL CHEMISTRY 277 (3) 1878 - 1883 0021-9258 2002/01 [Refereed][Not invited]
     
    Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O-2] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O-2)beta(Fe-O-2)][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O-2 binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O-2 equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O-2-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2) were measured by using an Imai apparatus. It was shown that the first and second O-2 equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg-1, respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O-2 binding study.
  • N Shibayama, S Saigo
    FEBS LETTERS 492 (1-2) 50 - + 0014-5793 2001/03 [Refereed][Not invited]
     
    The main features of cooperative oxygenation human hemoglobin have been described by assuming the equilibrium between two affinity conformations of the entire molecule, T and R, However, the molecular basis for explaining the wide variation in the O-2 affinities of the deoxy T state has remained obscure, We address this long-standing issue by trapping the conformational states of deoxyhemoglobin molecules within wet porous transparent silicate sol-gels. The equilibrium O-2 binding measurements of the encapsulated deoxyhemoglobin samples showed that deoxyhemoglobin free of anions coexists in two conformations that differ in O-2 affinity by 40 times or more, and addition of inositol hexaphosphate to this anion-free deoxyhemoglobin brings about a very slow redistribution of these affinity conformations, These results are the first, direct demonstration of the existence of equilibrium between two (at least two) functionally distinguishable conformational states in the T state deoxyhemoglobin. (C) 2001 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.
  • SHIBAYAMA Naoya
    Biophysics 40 (5) 346 - 349 0582-4052 2000/09 [Not refereed][Not invited]
  • G Miyazaki, H Morimoto, KM Yun, SY Park, A Nakagawa, H Minagawa, N Shibayama
    JOURNAL OF MOLECULAR BIOLOGY 292 (5) 1121 - 1136 0022-2836 1999/10 [Refereed][Not invited]
     
    Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha(2)(Fe)beta(2)(M) and alpha(2)(M)beta(2)(Fe); M = Mg(II),Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha(2)(Fe)beta(2)(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effecters, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha(2)(Fe)beta(2)(Mg) and alpha(2)(Fe-CO)beta(2)(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K-1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effecters is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution. We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins. (C) 1999 Academic Press.
  • Naoya Shibayama
    Journal of Molecular Biology 285 (4) 1383 - 1388 0022-2836 1999/01 [Refereed][Not invited]
     
    A controversy still exists over whether the molecular basis of hemoglobin cooperativity can be more appropriately explained by one of two classic allosteric models, the concerted and sequential models. To distinguish these two models from the viewpoint of their fundamental processes, namely, the presence or absence of conformational equilibria, we have trapped the conformations of nickel(II)-iron(II) hybrid hemoglobin molecules with two CO-bound, α2(Fe-CO)β2(Ni) and α2(Ni)β2(Fe-CO), by encapsulation in the water-filled pores of sol-gel-derived transparent silica-gels. In our experimental system, nickel(II) protoporphyrin binds neither O2 nor CO and mimics a fixed deoxyheme, and the gel matrix provides a means of inhibiting large-scale protein structural changes, thus enabling O2 equilibrium study of the hybrids still in their doubly liganded conformations. Results showed that two conformations of widely different O2 affinity exist together in each doubly liganded hemoglobin, providing a direct proof of the concerted mechanism versus the sequential mechanism.
  • N Shibayama, S Saigo
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 121 (2) 444 - 445 0002-7863 1999/01 [Refereed][Not invited]
  • S Unzai, R Eich, N Shibayama, JS Olson, H Morimoto
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 (36) 23150 - 23159 0021-9258 1998/09 [Refereed][Not invited]
     
    Despite a large amount of work over the past 30 years, there is still no universal agreement on the differential reactivities of the individual alpha and beta subunits in human hemoglobin. To address this question systematically, we prepared a series of hybrid hemoglobins in which heme was replaced by chromium(III), manganese(III), nickel(II), and magnesium(II) protoporphyrin IXs in either the alpha or beta subunits to produce alpha(2)(M)beta(2)(Fe)(1) and alpha(2)(Fe)beta(2)(M) tetramers. None of the abnormal metal complexes react with dioxygen or carbon monoxide. The O-2 affinities of the resultant hemoglobins vary from 3 mu M-1 (Cr(III)/Fe(II) hybrids) to 0.003 mu M-1 (Mg(II)/Fe(II) hybrids), covering the full range expected for the various high (R) and low (T) affinity quaternary conformations, respectively, of human hemoglobin A(0). The alpha and beta subunits in hemoglobin have similar O-2 affinities in both quaternary states, despite the fact that the R to T transition causes significantly different structural changes in the alpha and beta heme pockets, This functional equivalence almost certainly evolved to maintain high n values for efficient O-2 transport.
  • N Shibayama, H Morimoto, S Saigo
    BIOCHEMISTRY 37 (18) 6221 - 6228 0006-2960 1998/05 [Refereed][Not invited]
     
    A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha(+CN-)beta(+CN-))(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad, Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha(+CN-)beta(+CN-))(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N-2 With HCN. This confirms our view that previous experimental data on (alpha(+CN-)beta(+CN-))(alpha beta) Obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha(+CN-)beta(+CN-))(alpha beta) (i.e., greater than or equal to -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha(+CN-)beta(+CN-))(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange, These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.
  • AONO OSAMU, SAIGO SATOSHI, HARADA MITSUO, SHIBAYAMA NAOYA
    物理教育 45 (6) 328 - 329 0385-6992 1997/12 [Refereed][Not invited]
  • N Shibayama, H Morimoto, S Saigo
    BIOCHEMISTRY 36 (15) 4375 - 4381 0006-2960 1997/04 [Refereed][Not invited]
     
    It has been reported that hybridization of the equimolar mixture of cyanomethemoglobin and deoxyhemoglobin through dimer exchange reaction results in establishment of an approximately binomial (1:2:1) equilibrium distribution of these parental hemoglobins and their hybrid molecule, (alpha(+CN-)beta(+CN-))-(alpha beta), within several days under anaerobic conditions at pH 7.4, 21.5 degrees C, leading to a hyper (i.e., about 170 times) thermodynamic stability of (alpha(+CN-)beta(+CN-))(alpha beta) relative to the stability of the other diliganded species at pH 7.4, 21.5 degrees C [Daugherty, M. A., Shea, M. A., Johnson, J. A., LiCata, V. J., Turner, G. J., & Ackers, G. K. (1991) Proc. Natl. Acad. Sci, U.S.A. 88, 1110-1114], To examine whether the published ''binomiality'' for this deoxy-cyanomet hybrid system really reflects the thermodynamic stability of (alpha(+CN-)-beta(+CN-))(alpha beta), we used a binomial. (1:2:1) equilibrium distribution of the equimolar mixture of cyanomethemoglobin and fully oxygenated hemoglobin as a starting condition, and then this system was rapidly deoxygenated. We found that the relative population of the hybrid was reduced to 8.6% of the total upon deoxygenation. It was also found that the hybridization experiment under anaerobic conditions was not allowed to continue for a long time due to a valency exchange reaction between deoxy and cyanomet derivatives. For instance, a 48 h incubation resulted in the oxidation of 44% of Fe2+ to Fe3+ hemes in the original deoxyhemoglobin and the reduction of 42% of Fe3+ to Fe2+ hemes in the original cyanomethemoglobin. These results suggest that a real distribution of the deoxy-cyanomet hybrid system at equilibrium is fairly far from 1:2:1 (binomial distribution), and the thermodynamic stability of (alpha(+CN-)beta(+CN-))(alpha beta) is less than one-tenth of the hyperstability previously reported. In addition, most of the previous results on deoxy-cyanomet valency hybrids placed under long anaerobic conditions should be subject to reexamination due to possible valency exchange reactions.
  • S Unzai, H Hori, G Miyazaki, N Shibayama, H Morimoto
    JOURNAL OF BIOLOGICAL CHEMISTRY 271 (21) 12451 - 12456 0021-9258 1996/05 [Not refereed][Not invited]
     
    Cr(III)-Fe(II) hybrid hemoglobins, alpha(2)(Cr)beta(2)(Fe) and alpha(2)(Fe)beta(2)(Cr), in which hemes in either the alpha- or beta-subunits were substituted with chromium(III) protoporphyrin IX (Cr(III)PPIX), were prepared and characterized by oxygen equilibrium measurements. Because Cr(III)PPM binds neither oxygen molecules nor carbon monoxide, the oxygen equilibrium properties of Fe(II) subunits within these hybrids can be analyzed by a two-step oxygen equilibrium scheme. The oxygen equilibrium constants for both hybrids at the second oxygenation step agree with those for human adult hemoglobin at the last oxygenation step (at pH 6.5-8.4 with and without inositol hexaphosphate at 25 degrees C). The similarity between the effects of the Cr(III)PPM and each subunits' oxyheme on the oxygen equilibrium properties of the counterpart Fe(II) subunits within hemoglobin indicate the utility of Cr(III)PPM as a model for a permanently oxygenated heme within the hemoglobin molecule. We found that Cr(III)-Fe(II) hybrid hemoglobins have several advantages over cyanomet valency hybrid hemoglobins, which have been frequently used as a model system for partially oxygenated hemoglobins. In contrast to cyanomet heme, Cr(III)PPM within hemoglobin is not subject to reduction with dithionite or enzymatic reduction systems. Therefore, we could obtain more accurate and reasonable oxygen equilibrium curves of Cr(III)-Fe(II) hybrids in the presence of an enzymatic reduction system, and we could obtain single crystals of deoxy-alpha(2)(Cr)beta(2)(Fe) when grown in low salt solution in the presence of polyethylene glycol 1000 and 50 mM dithionite.
  • N SHIBAYAMA, T YONETANI, RM REGAN, QH GIBSON
    BIOCHEMISTRY 34 (45) 14658 - 14667 0006-2960 1995/11 [Refereed][Not invited]
     
    The geminate and bimolecular binding of CO, O-2, and NO to [alpha-Ni(II)](2)-[beta-Fe(II)](2) and [alpha-Fe(II)](2)-[beta-Ni(II)](2) hybrid hemoglobins has been studied. Bimolecular reactions: At pH 6.6 and 20 degrees both hybrids bind CO at 0.15 x 10(6) M(-1) s(-1). Reactions with oxygen: At pH 6.6 the on rates are 4.8 and 7.5 x 10(6) M(-1) s(-1) for alpha- and beta-hybrids, respectively; the off rate is approximately 2 x 10(3) s(-1) for both. At pH 8 the alpha-Fe hybrid shows cooperativity whereas the beta-hybrid does not. Nanosecond geminate reactions: Faster bimolecular rates correlate with larger, geminate amplitudes; thus alpha-Fe hybrids have larger amplitudes, and O-2 geminate amplitudes are larger than these with CO. At pH 8, 50% of O-2 recombines with the alpha-hybrid. With NO, nanosecond geminate recombination is observable only with the beta-hybrid. Picosecond reactions: alpha-Hybrids show picosecond recombination of O-2. With NO, alpha-hybrids recombine at 30 ns(-1), beta-hybrids at 0.3 ns(-1). The NO picosecond rates correlate with the molecular dynamics which shows ligands leaving the beta-Fe atom early and regularly, but remaining near the alpha-Fe atom. The results may be explained by assuming an interaction between the alpha-subunits giving rise to a high-affinity fast-reacting form, whereas the beta-subunits only become fast-reacting when an R-T conformation change analogous to that of hemoglobin A takes place. A third allosteric state is postulated to explain the results.
  • N SHIBAYAMA, M IKEDASAITO, H HORI, K ITAROKU, H MORIMOTO, S SAIGO
    FEBS LETTERS 372 (1) 126 - 130 0014-5793 1995/09 [Refereed][Not invited]
     
    Copper(II)-iron(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunits are substituted with copper(II protoporphyrin IX, have been prepared, The affinities of the ferrous-subunits in both hybrids for the first binding oxygen are as low as the affinity of deoxyhemoglobin under various solution conditions, indicating the equality of behavior in copper(II) protoporphyrin IX and deoxyheme, Electron paramagnetic resonance (EPR) examinations on these hybrids at room temperature show that the interaction between copper(II) and the proximal histidine (F8) is specifically weakened in the alpha subunits within a low affinity conformation of hemoglobin, These results suggest that copper(II) protoporphyrin IX is a useful EPR probe at room temperature for investigating the deoxyheme environment in hemoglobin.
  • N SHIBAYAMA, S SAIGO
    JOURNAL OF MOLECULAR BIOLOGY 251 (2) 203 - 209 0022-2836 1995/08 [Refereed][Not invited]
     
    We have used the sol-gel method to encapsulate oxy- and deoxy haemoglobins in transparent wet porous silica gels and fixed their original functional states with the retention of the reversible oxygenation properties as well as the intact spectroscopic properties. Haemoglobin originally encapsulated in aerobic gel binds oxygen non-cooperatively with very high affinity, corresponding to that for the last oxygen molecule binding to haemoglobin in solution. In contrast, haemoglobin originally encapsulated in anaerobic gel binds oxygen non-cooperatively with very low affinity, comparable to that for the first oxygen molecule binding to haemoglobin in solution. Furthermore, a detailed comparison of visible absorption spectra of deoxygenated haemoglobins originally encapsulated in aerobic and anaerobic gels indicates the retention of their original quaternary structures during the oxygenation or deoxygenation process. These results demonstrate that oxygen affinities of oxy- and deoxyhaemoglobins in solution can be satisfactorily fixed by encapsulation in wet porous silica gels, which presumably prevents the changes in the quaternary structures of haemoglobin. In addition, these results suggest a new capability of the sol-gel method to control the structural states of a variety of proteins, and further open up a new area of investigation of protein structure-function relationships. (C) 1995 Academic Press Limited
  • AONO OSAMU, SAIGO SATOSHI, HARADA MITSUO, SHIBAYAMA NAOYA
    物理教育 42 (3) 307 - 310 0385-6992 1994/09 [Refereed][Not invited]
  • S SAIGO, H HASHIMOTO, N SHIBAYAMA, M NOMURA, T NAGAMURA
    BIOCHIMICA ET BIOPHYSICA ACTA 1202 (1) 99 - 106 0006-3002 1993/09 [Refereed][Not invited]
     
    The reaction of cyanide metmyoglobin (Mb+CN-) with dithionite produces a transient intermediate, supposed to be cyanide-ligated ferrous myoglobin. The Fe K-edge X-ray absorption spectrum of the intermediate has been measured by using rapid freezing and compared with those of Mb+CN- and deoxymyoglobin (deoxyMb). The shapes of the XANES (X-ray Absorption Near Edge Structure) spectra of Mb+CN- and the intermediate are very similar, including the intensity ratios of the peak C1 to D. This indicates that CN- remains bound with a linear Fe-C-N configuration in the intermediate. The absorption edge of the intermediate is shifted to 1.2 eV lower energy than that of Mb+CN-, reflecting a valence change in the heme iron. The EXAFS (Extended X-ray Absorption Fine Structure) spectrum of the intermediate closely resembles that of Mb+CN- but significantly differs from that of deoxyMb. Analysis shows that the average iron-nearest neighbor atom distance is 1.99 +/- 0.01 angstrom for both Mb+CN- and the intermediate and 2.05 +/- 0.01 angstrom for deoxyMb. These results imply that the local structure around the heme iron of Mb+CN- does not change upon reduction until the cyanide ligand is released.
  • N SHIBAYAMA, K IMAI, H MORIMOTO, S SAIGO
    BIOCHEMISTRY 32 (34) 8792 - 8798 0006-2960 1993/08 [Refereed][Not invited]
     
    Asymmetric Ni(II)-Fe(II) hybrid hemoglobin, XL[alpha(Fe)beta(Fe)][alpha(Ni)beta(Ni)], in which the alpha1beta1 dimer containing ferrous protoporphyrin IX and the complementary alpha2beta2 dimer containing Ni(II) protoporphyrin IX were cross-linked between Lys-82beta1 and Lys-82beta2 by reaction with bis(3,5-dibromosalicyl) fumarate, was synthesized and characterized. We have previously shown that (i) Ni(II) protoporphyrin IX, which binds neither oxygen nor carbon monoxide, mimics a fixed deoxyheme with respect to its effect on the oxygen equilibrium properties of the counterpart iron subunits in both symmetric Ni(II)-Fe(II) hybrid Hbs [Shibayama, N., Morimoto, H., & Miyazaki, G. (1986) J. Mol. Biol. 192, 323-329] and (ii) the cross-linking used in this study little affects the oxygen equilibrium properties of hemoglobin [Shibayama, N., Imai, K., Hirata, H., Hiraiwa, H., Morimoto, H., & Saigo, S. (1991) Biochemistry 30,8158-8165]. These remarkable features of our model allowed us to measure the oxygen equilibrium curves for the first two steps of oxygen binding to the alpha1beta1 dimer within the hemoglobin tetramer. At all pH values examined, the affinities of this asymmetric hybrid for the first oxygen molecule are as low as those of native hemoglobin. The hybrid did not show cooperative oxygen binding at pH 6.4, while significant cooperativity was observed with rising pH; i.e., the Hill coefficient was increased from 1.41 to 1.53 upon a pH change from 7.4 to 8.4. The electronic absorption spectrum of Ni(II) protoporphyrin IX in the alpha2 subunit was changed upon carbon monoxide (or oxygen) binding to the alpha1beta1 dimer. This change provides additional information about the interaction between liganded and unliganded dimers within the asymmetric tetramer.
  • Rapid-freeze XAFS characterization of kinetic intermediates of metalloproteins
    Saigo, S, Hashimoto, H, Shibayama, N, Nomura, M, Nagamura, T
    Jpn. J. Appl. Phys. 32 538 - 540 1993 [Refereed][Not invited]
  • K ADACHI, J KIM, N SHIBAYAMA
    BIOCHIMICA ET BIOPHYSICA ACTA 1079 (3) 268 - 272 0006-3002 1991/09 [Refereed][Not invited]
     
    Polymerization of half-liganded Hb S was investigated using Ni(II)-Fe(II) hybrid Hb S, in which heme in either alpha or beta-s subunits is replaced by Ni (II) protoporphyrin IX. Studies on the polymerization of these hybrid hemoglobins were carried out under aerobic conditions. Both alpha-2(Ni)beta-2-s(Fe-CO) and alpha-2(Fe-CO)beta-2-s(Ni) polymerized with a distinct delay time as do native deoxy-Hb S and Ni(II) Hb S. However, the critical concentration for polymerization of half-liganded Hb S, alpha-2(Ni)beta-2-s(Fe-CO) and alpha-2(Fe-CO)beta-2-s (Ni), was 4- and 8-times higher, respectively, than that of Ni(II)-Hb S. Kinetics of polymerization of both deoxygenated hybrid hemoglobins with CO completely removed were the same, although the critical concentrations for polymerization were intermediate between those for deoxy-Hb S and Ni(II)-Hb S. These results suggest that the small tertiary conformational change associated with the doubly liganded state may be much less favorable to polymerization than the completely unliganded state of Hb S. The conformational change depends on whether alpha or beta chain is liganded. The ease of polymerization and low solubility of sickle hemoglobin is dependent not only on quaternary, but on tertiary structural changes, as well as on the substitution of Val for Glu at the beta-6 position.
  • N SHIBAYAMA, K IMAI, H HIRATA, H HIRAIWA, H MORIMOTO, S SAIGO
    BIOCHEMISTRY 30 (33) 8158 - 8165 0006-2960 1991/08 [Refereed][Not invited]
     
    We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82-beta-1 and lysine-82-beta-2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93-beta-1 toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.
  • B LUISI, B LIDDINGTON, G FERMI, N SHIBAYAMA
    JOURNAL OF MOLECULAR BIOLOGY 214 (1) 7 - 14 0022-2836 1990/07 [Refereed][Not invited]
  • B LUISI, N SHIBAYAMA
    JOURNAL OF MOLECULAR BIOLOGY 206 (4) 723 - 736 0022-2836 1989/04 [Refereed][Not invited]
  • K ADACHI, J KIM, N SHIBAYAMA
    SICKLE CELL DISEASE / 565 356 - 357 1989 [Refereed][Not invited]
  • N SHIBAYAMA, T INUBUSHI, H MORIMOTO, T YONETANI
    BIOCHEMISTRY 26 (8) 2194 - 2201 0006-2960 1987/04 [Refereed][Not invited]
  • 混成ヘモグロビンの協同作用
    森本英樹, 柴山修哉, 宮崎源太郎
    蛋白質・核酸・酵素 32 (6) 557 - 565 1987 [Refereed][Not invited]
  • N SHIBAYAMA, H MORIMOTO, G MIYAZAKI
    JOURNAL OF MOLECULAR BIOLOGY 192 (2) 323 - 329 0022-2836 1986/11 [Refereed][Not invited]
  • Shibayama, N, Morimoto, H, Kitagawa, T
    Journal of Molecular Biology 192 330 - 336 1986 [Refereed][Not invited]

Conference Activities & Talks

MISC

  • SY Park, N Shibayama, T Hiraki, JRH Tame  BIOCHEMISTRY  43-  (27)  8711  -8717  2004/07  [Not refereed][Not invited]
     
    A number of ligand binding studies of human adult hemoglobin (HbA) cross-linked between Lys 82beta(1) and Lys 82beta(2) with bis(3,5-dibromosalicyl)fumarate have been reported. The oxygen binding properties of native HbA, including the cooperativity and Bohr effect, are not substantially changed by the modification, provided care is taken to remove electrophoretically silent impurities arising from side reactions. We have refined the high-resolution structure of this modified Hb and found it adopts the T state when crystallized in the absence of heme ligands, contrary to a previously published structure. These results suggest the slightly altered crystal form determined previously may be due to unremoved side products of the cross-linking reaction with high oxygen affinity. Two nickel-substituted Hbs cross-linked in the same way have also been crystallized in the presence of carbon monoxide, which binds only to the ferrous heme. In the case of the nickel-substituted alpha subunit, the absence of a covalent link between the central metal of the heme and the proximal histidine leads to a new conformation of the histidine stabilized by a water molecule. This structure may mimic that of partially NO-liganded species of HbA; however, overall, the changes are highly localized, and both doubly ligated species are in the T conformation.
  • S Saigo, N Shibayama  BIOCHEMISTRY  42-  (32)  9669  -9676  2003/08  [Not refereed][Not invited]
     
    Theory and simulations predict that the folding kinetics of protein-like heteropolymers become nonexponential and glassy (i.e., controlled by escape from different low-energy misfolded states) at low temperatures, but there was little experimental evidence for such behavior of proteins. We have developed a stopped-flow instrument working reliably down to -40 degreesC with high mixing capability and applied it to study the refolding kinetics of horse cytochrome c (cyt c) and hen egg white lysozyme at temperatures below 0 degreesC in the presence of antifreeze NaCl, LiCl, or ethylene glycol and above 0 degreesC in the presence and absence of antifreeze. The refolding was initiated by rapid dilution of the guanidine hydrochloride unfolded proteins, and the kinetics were monitored by intrinsic tryptophan fluorescence. Highly nonexponential kinetics extended over 3 decades in time (0.01-10 s) were observed in the early phases of the refolding of cyt c and lysozyme in the temperature range of -35 to 5 degreesC. These results are in agreement with the theoretical prediction, suggesting that the folding energy landscapes of these proteins are rugged in the upper portions.
  • S Adachi, SY Park, JRH Tame, Y Shiro, N Shibayama  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA  100-  (12)  7039  -7044  2003/06  [Not refereed][Not invited]
     
    Human Hb, an alpha(2)beta(2) tetrameric oxygen transport protein that switches from a T (tense) to an R (relaxed) quaternary structure during oxygenation, has long served as a model for studying protein allostery, in general. Time-resolved spectroscopic measurements after photodissociation of CO-liganded Hb have played a central role in exploring both protein dynamical responses and molecular cooperativity, but the direct visualization and the structural consequences of photodeligation have not yet been reported. Here we present an x-ray study of structural changes induced by photodissociation of half-liganded T-state and fully liganded R-state human Hb at cryogenic temperatures (25-35 K). On photodissociation of CO, structural changes involving the heme and the F-helix are more marked in the a subunit than in the beta subunit, and more subtle in the R state than in the T state. Photodeligation causes a significant sliding motion of the T-state beta heme. Our results establish that the structural basis of the low affinity of the T state is radically different between the subunits, because of differences in the packing and chemical tension at the hemes.
  • N Shibayama, S Saigo  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY  125-  (13)  3780  -3783  2003/04  [Not refereed][Not invited]
     
    A comparison of the O-2 equilibrium curves of sperm-whale myoglobin locked in the liganded (CO-bound) and unliganded (deoxy) conformations by encapsulation in a wet porous sol-gel silica reveals a marked difference between them. The CO-bound state locked myoglobin showed a nearly monophasic (hyperbolic) O-2 equilibrium curve with a dissociation constant of 0.2 Torr, which is smaller than that of myoglobin in solution (0.5 Torr). On the other hand, the deoxy state-locked myoglobin exhibited a multiphasic O-2 equilibrium curve that can be represented by a sum of three independent components with dissociation constants of 0.19, 0.90, and 44 Torr, respectively, indicating that deoxymyoglobin exists in multiple conformations. These results show that myoglobin can be frozen into ligand-dependent conformational populations at room temperature in the wet sol-gel and suggest that the overall O-2 equilibrium properties of myoglobin in solution are generated by a redistribution of protein conformational populations in response to ligand binding.
  • Differences in changes of the α1β2 subnnit contacts between Ligand Binding to the α and β subnnits of Hemoglobin A(共著)
    Biochemistry  41, 10010-10020-  2002  [Not refereed][Not invited]
  • Journal of Biological Chemistry  277, 38791-38796-  2002  [Not refereed][Not invited]
  • KM Yun, H Morimoto, N Shibayama  JOURNAL OF BIOLOGICAL CHEMISTRY  277-  (3)  1878  -1883  2002/01  [Not refereed][Not invited]
     
    Considerable controversy remains as to the functional and structural properties of the asymmetric alpha1beta1 half-oxygenated intermediate of human hemoglobin, consisting of a deoxygenated and an oxygenated dimer. A recent dimer-tetramer equilibrium study using [Zn(II)/Fe(II)-O-2] hybrid hemoglobins, in which Zn-protoporphyrin IX mimics a deoxyheme, showed that the key intermediate, [alpha(Fe-O-2)beta(Fe-O-2)][alpha(Zn)beta(Zn)], exhibited an enhanced tetramer stability relative to the other doubly oxygenated species. This is one of the strongest findings in support of distinctly favorable intra-dimer cooperativity within the tetramer. However, we present here a different conclusion drawn from direct O-2 binding experiments for the same asymmetric hybrid, [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)], and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2). In this study, the O-2 equilibrium curves for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] were determined by an O-2-jump stopped-flow technique to circumvent the problem of dimer rearrangement, and those for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2) were measured by using an Imai apparatus. It was shown that the first and second O-2 equilibrium constants for [alpha(Fe)beta(Fe)][alpha(Zn)beta(Zn)] are 0.0209 mmHg(-1) and 0.0276 mmHg-1, respectively, that are almost identical to those for [alpha(Fe)beta(Zn)](2) or [alpha(Zn)beta(Fe)](2). Therefore, we did not observe large difference among the asymmetric and symmetric hybrids. The discrepancy between the present and previous studies is mainly due to previously observed negative cooperativity for [alpha(Fe)beta(Zn)](2) and [alpha(Zn)beta(Fe)](2), which is not the case in our direct O-2 binding study.
  • FEBS Letters  492, 50-53-  2001  [Not refereed][Not invited]
  • Journal of Molecular Biology  285, 1383-1388-  1999  [Not refereed][Not invited]
  • Journal of the American Chemical Society  121, 444-445-  1999  [Not refereed][Not invited]
  • Journal of Biological Chemistry  273, 23150-23159-  1998  [Not refereed][Not invited]
  • Asymmetric cyanomet valency hybrid hemoglobin, (α+CN-β+CN-)(αβ) : The issue of valency exchange.
    Biochemistry  3718, 6221-6228-  1998  [Not refereed][Not invited]
  • Journal of Biological Chemistry  271, 12451-12456-  1996  [Not refereed][Not invited]
  • Naoya Shibayama, Satoshi Saigo  Journal of Molecular Biology  251-  (2)  203  -209  1995/08  [Not refereed][Not invited]
     
    We have used the sol-gel method to encapsulate oxy- and deoxy haemoglobins in transparent wet porous silica gels and fixed their original functional states with the retention of the reversible oxygenation properties as well as the intact spectroscopic properties. Haemoglobin originally encapsulated in aerobic gel binds oxygen non-cooperatively with very high affinity, corresponding to that for the last oxygen molecule binding to haemoglobin in solution. In contrast, haemoglobin originally encapsulated in anaerobic gel binds oxygen non-cooperatively with very low affinity, comparable to that for the first oxygen molecule binding to haemoglobin in solution. Furthermore, a detailed comparison of visible absorption spectra of deoxygenated haemoglobins originally encapsulated in aerobic and anaerobic gels indicates the retention of their original quaternary structures during the oxygenation or deoxygenation process. These results demonstrate that oxygen affinities of oxy- and deoxyhaemoglobins in solution can be satisfactorily fixed by encapsulation in wet porous silica gels, which presumably prevents the changes in the quaternary structures of haemoglobin. In addition, these results suggest a new capability of the sol-gel method to control the structural states of a variety of proteins, and further open up a new area of investigation of protein structure-function relationships. © 1995 Academic Press Limited.
  • Biochemistry  34, 14658-14667-  1995  [Not refereed][Not invited]
  • Biochemistry  32,8792-8798-  1993  [Not refereed][Not invited]
  • Polymerization and solubility of Ni(II)-Fe(II) hybid HbS. (共著)
    Biochimicu et Biophysica Acta  1079, 268-272-  1991  [Not refereed][Not invited]
  • Journal of Molecular Biology  214, 7-14-  1990  [Not refereed][Not invited]
  • Polymerization of partially liganded hemoglobin S. (共著)
    Sickle Cell Disease  565, 356-357-  1989  [Not refereed][Not invited]
  • Journal of Molecular Biology  206, 723-736-  1989  [Not refereed][Not invited]
  • Properties of chemically modified Ni(II)-Fe(II) hybrid hemoglobins.
    Journal of Molecular Biology  192, 330-336-  1986  [Not refereed][Not invited]
  • Journal of Molecular Biology  192, 323-329-  1986  [Not refereed][Not invited]

Research Grants & Projects

  • 働く最中のヘモグロビン分子内動態の原子レベル追跡
    文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2016/04 -2019/03 
    Author : 柴山 修哉
  • バイオロジーにおける3D活性サイト科学
    文部科学省:科学研究費補助金(新学術領域研究)
    Date (from‐to) : 2014/07 -2019/03 
    Author : 佐々木 裕次
  • ヘモグロビンの四次構造変化を許容する結晶を用いた時分割構造解析法の開拓
    文部科学省:科学研究費補助金(新学術領域研究)
    Date (from‐to) : 2015/04 -2017/03 
    Author : 柴山 修哉
  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2011 -2013 
    Author : 柴山 修哉
     
    蛋白質分子の機能を調節している構造変化は、ヘモグロビンの事例を下敷きとしてT(tense)状態とR(relaxed)状態の2状態の平衡で説明されることが多い。しかし、肝心のヘモグロビンでは2状態モデル的な理解が成功していないのが実状である。また、ヘモグロビンの既知の結晶構造と溶液の酸素平衡機能データを組み合わせても、その分子メカニズムの本質に迫れない慢性的な手詰まり状態がここ30年以上も続いている。 平成23年度の研究では、ヘモグロビンの既知の結晶構造、特に4次構造の異なる二つのリラックス状態(R状態とR2状態)の機能を結晶の酸素平衡曲線から直接決定した。実験では、安定なRとR2結晶を得るため、分子表面に一残基変異を持つ異常ヘモグロビンC[β6Glu→Lys]を用いた。ヘモグロビンCの特筆すべき特徴は、酸素平衡機能は通常のヘモグロビンAと同じであるが、溶解度が著しく低いおかげで極めて良質の結晶が速やかに得られる点にある。結晶の酸素平衡曲線は、現有の顕微分光光度計、精密ガス混合機、酸素濃度計を組み合わせたシステムを構築し測定した。今回の実験結果から、RとR2は酸素親和性の異なる独立なアロステリック状態ではなく、酸素親和性最高のアロステリック状態(R)の構造多形性の反映であることが明らかになった。 更にこの年度は、酸素親和性がTとRの中間的なアロステリック状態(P状態)の結晶化に用いる最適試料を選択するため、ヘモグロビンの4個のヘムFe(II)のうち2個を酸素(またはCO)と結合しないNi(II)で置き換えた4種類の金属置換混成ヘモグロビンを調製し、COが2個結合した中間状態ヘモグロビン4種類の溶液での性質を比較した。これらの結果をもとに、α1とβ2サブユニットにCOが2個結合した中間状態の結晶化を系統的に行い、P状態の構造解析に有望な結晶化条件を見つけ出した。
  • 文部科学省:科学研究費補助金(萌芽研究, 挑戦的萌芽研究)
    Date (from‐to) : 2008 -2009 
    Author : 柴山 修哉, 朴 三用
     
    1. 初期状態EA4のX線結晶構造解析EA4はカイコ休眠卵の休眠打破に必要な冬期低温持続時間(5℃で約2週間)を計る機能を持つタイマータンパク質であり、室温の休眠卵中では時間読み停止機能を持つ38残基ペプチド(PIN)との複合体を形成することで時間読み開始前の初期状態に凍結されると報告されている。このような情報をもとに、カイコサナギ個体を宿主としたEA4とPINの共発現系を構築し結晶化スクリーニングを集中的に行った。しかし、複合体の構造解析にはまだ成功していない。これには理由がある。先ず、カイコ個体中ではPINが酵素分解を受け雑多な部分配列ペプチド集合体となり試料を複雑にしていること。また、試料中にPIN以外の未知のEA4結合性ペプチドが混在し、EA4・PIN複合体の形成効率を低下させていることである。本年度は、粗精製した共発現試料に対して大腸菌発現で作製したPINを大過剰に加え、25℃での限外濾過(分画分子量1万)を繰り返すことにより雑ペプチドを除去する方法を新たに取り入れた。ごく最近、この方法で精製した試料の微小結晶を得ることに成功した。現在、X線回折実験に適した大型単結晶の作製条件を検討している。また本年度は、上述の未知のEA4結合性ペプチドの全48アミノ酸配列を決定した。2. 計時現象の計算機シミュレーションEA4の時間読みは、低温刺激によりPINが解離することで始まり、10日以上の遅延相の後に一過性の鋭いATPase活性を発現することで終了する。この計時現象を説明する最も有力なモデルは、EA4分子内の銅イオンが配位子交換反応を経ながら活性中心までゆっくり移動し活性型に転移する「多段階逐次モデル」である。本年度は、このモデルの妥当性を計算機シミュレーションで検討した。その結果、実際のデータを再現するためには60以上の異なる段階を仮定する必要があることが分かった。これはかなり無理のある状態数であり、モデルの修正を検討中である。
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2005 -2007 
    Author : Naoya SHIBAYAMA
     
    Entrapment of proteins in wet, optically transparent, porous silica gel matrices has enabled a dramatic expansion of the folding time, allowing direct observation of the entire folding pathway using spectroscopic techniques. In this study, we have focused on the folding reactions of the following three model proteins.1. Horse cytochrome cIn wet silica gels, cytochrome c folds over a period of hours, days, weeks, or longer, depending on the aging time. During refolding in silica gels, collapse and helix formation occur in a stepwise manner, as observed in aqueous solution. Analysis of kinetics and transient spectra reveals a sequence of four distinct intermediates with progressively increasing degree of folding, two of which closely resemble those previously characterized in solution, namely, the early collapsed and the molten globule intermediates. The other two are the pre-collapsed and pre-molten globule intermediates that may escape detection by conventional kinetic methods.2. Bovine ubiquitinWhile ubiquitin folds in an apparent two-state manner in aqueous solution, a partially structured intermediate is populated on the folding pathway of ubiquitin when entrapped in wet silica gels. This finding suggests an interesting possibility that our approach is able to stabilize and capture on-pathway "hidden" folding intermediates that may exist on the folding pathway but escape detection by conventional techniques.3. Bovineβ-lactoglobulinThe refolding of β-lactoglobulin was also very slow in wet silica gels. Analysis of the transient CD spectra allows us to probe the early folding events that consist of non-native a-helix formation, native β-sheet core formation, and further a-helix to (β-sheet transition. These results illustrate the potential usefulness of our approach for high-resolution analysis of rapid folding events without significantly perturbing the folding pathway.
  • 文部科学省:科学研究費補助金(萌芽的研究, 萌芽研究)
    Date (from‐to) : 2000 -2002 
    Author : 柴山 修哉
     
    1.ミオグロビンのコンホメーションの固定:ミオグロビンの脱酸素状態と一酸化炭素結合状態をウエットなゾル・ゲル中に固定し,これら二つのコンホメーションの違いを酸素平衡機能の観点から調べた.一酸化炭素結合状態で固定されたミオグロビンは,ほぼ均一な酸素平衡曲線を示し,その酸素親和性(0.2mmHg)は溶液のミオグロビンの親和性(0.5mmHg)より約2倍高かった.一方,脱酸素状態で固定されたミオグロビンの酸素平衡曲線は不均一性を示し,酸素親和性の大幅に異なる3成分(0.19,0.9,44mmHgの3成分)の和で記述できることが分かった.これらの結果は,ミオグロビンの各状態中に存在するコンホメーションの分布を室温のゾル・ゲルで凍結できることを示しており,一見単純にみえる溶液中ミオグロビンの酸素平衡特性は,リガンド結合に伴うコンホメーション分布の再編から生まれることを示唆している.2.ゾル・ゲル中シトクロムcのrefolding:薄いフイルム状のゾル・ゲルに閉じこめたネイティブ状態のシトクロムcがpH1.5で酸変性する過程と,pH4.5で再び折れ畳まれる過程をヘムの光吸収スペクトル変化で追跡した.ゾル・ゲル中シトクロムcのrefolding過程は,通常の溶液中のそれと比較して数桁から10桁のオーダーで減速することが示された.特に,重合度を高くしたゾル・ゲル中ではrefoldingの中間体(いわゆるモルテングロビュール状態)を室温で長時間(数時間)捕捉することができた.3.ゾル・ゲル固定法で見つかった"酸素親和性の高いT状態ヘモグロビン"のX線構造解析を意図して,いくつかの結晶試料について構造の精密化を行っている.現在のところ,まだ目的の構造は得られていない.
  • Study on the timer mechanism of the clock protein EA4
    Date (from‐to) : 2001
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1998 -1999 
    Author : 柴山 修哉
     
    ヘモグロビンの酸素結合には強い協同性がある。この協同現象は、2状態モデル的なT-R転移で説明される場合が多い。しかし、この考え方には疑問がある。ヒトヘモグロビンのR状態の酸素親和性は溶液条件によらずほぼ一定値をとるが、T状態の酸素親和性は溶液条件に依存して約100倍も変化してしまう。このT状態の酸素親和性変化の原因についてはまだなにも分かっていない。今回、この問題をはっきりさせるため、いくつかの溶液条件のT状態デオキシヘモグロビンの構造をゾル・ゲル包括で固定し、酸素平衡曲線を測定した。その結果、T状態ヘモグロビンの中に酸素親和性の大幅に異なる二種類の状態が存在することが分かった。即ち、T状態の酸素親和性の最低値を持つ状態と最高値を持つ状態が平衡で存在し、これらの存在比が変わることでT状態の酸素親和性変化が起こっているようである。ゾル・ゲル包括でとらえられた二種類のT状態の存在の裏付けをとるため以下の研究を行った。先ず、酸素親和性の低い色々なヘモグロビン(異常ヘモグロビンや金属置換混成ヘモグロビン)の溶液の酸素平衡曲線のデーターを再検討した。その結果、ヒトヘモグロビンの酸素親和性には共通の最低値があることが分かった。即ち、ヒトヘモグロビンには"最低親和性状態"が存在する。この状態が、ゲル中で見つかった"T状態の親和性の最低値を持つ状態"に対応する。また、この状態の酸素親和性は、デオキシヘモグロビン結晶の酸素親和性(結晶中では協同性は無い)と定量的に一致する。したがって、ヘモグロビンの"最低親和性状態"の構造は、デオキシヘモグロビンのX線結晶構造で与えられると考えるのが自然であろう。現在、ゲル中で見つかったもう一つの酸素親和性の高い方のT状態の構造的基盤を求めて研究を行っている。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1996 -1996 
    Author : 柴山 修哉
     
    1.多孔性ガラス中ヒトヘモグロビンの高次構造変化の速度を求める実験を以下の2つの方法で行った。(1)天然オキシヘモグロビンを多孔性ガラスに封入しデオキシ化して、デオキシヘムの光吸収変化からR→T変化速度を求めた。(2)酸素親和性がオキシ型とデオキシ型の中間になるようなニッケル・鉄混成ヘモグロビンを多孔性ガラス中に封入して温度ジャンプ法を行い、ニッケルポルフィリンの光吸収スペクトル変化からR→T及びT→R変化速度を求めた。これらの実験から、通常室温溶液中ではマイクロ秒領域にあるヘモグロビンの高次構造変化が、摂氏20度の多孔性ガラス中では数10時間のオーダーにまで遅くなっていることが分かった。また、構造変化の速度は摂氏35度では数倍速くなり、構造変化は各温度で可逆的であった。2.当初、連続測定型の酸素平衡曲線測定装置の開発を目指していたが、酸素分子のガラス中での拡散速度の問題と予算上の制約で実現できなかった。その代わり、比較的低酸素濃度まで測定できる不連続型のシステムを恒温プレハブ内に組み上げた。巨大ヘモグロビンのアロステリック単位を調べる実験は、このシステムを用いて1997年3月から開始する予定である。3.Xeフラッシュ照射後の多孔性ガラス中バクテリオロドプシン(紫膜)の光サイクルの挙動を分光学的に調べた。予想に反して、結果は通常のサスペンジョンでの実験結果とほぼ同じであった。バクテリオロドプシンの場合、膜から露出しているタンパク部分は少ないので、多孔性ガラスの構造同定作用が膜内部まで及ばなかったと考えている。現在、膜タンパク質の構造変化を抑える目的で、乾燥させたガラス中での固定化実験、及び、マルトース等の糖類を細孔中に入れて粘性をあげた系での固定化実験を試みている。
  • Slow motion analysis of protein dynamics within wet silica gels
    Date (from‐to) : 1995
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1994 -1994 
    Author : 柴山 修哉
     
    〈多孔性ガラスでヘモグロビンを安定に封入する技術開発〉、Ellerbyら(1992 Science 255,1113.)によって報告されたゾル-ゲル法を改良して、ヒトのオキシ型あるいはデオキシ型ヘモグロビンを可逆的酸素脱着能を保持したまま透明な多孔性ガラスに(リン酸緩衝液を細孔に含む状態のまま)封入することに成功した。この様にして初期状態をオキシ型あるいはデオキシ型で封入したヘモグロビンの光吸収スペクトルを通常の溶液中のスペクトルと比較したところ、両スペクトル共完全な一致が得られた。〈機能測定の結果〉初期状態をデオキシ型で封入したヘモグロビンとオキシ型で封入したヘモグロビンの酸素平衡機能を比較すると、両者共協同性は消失しており、酸素親和性の値は約500倍異なっていた。また、この時観測された酸素平衡定数は、通常のヘモグロビンに対する1個目及び4個目の酸素平衡定数とほぼ一致していたので、ヘモグロビン分子は封入時の高次構造を保持したまま安定な状態で多孔性ガラス中に存在し、リガンド脱着に伴う機能変化はほぼ完全に抑えられていることが結論された。〈構造測定の結果〉デオキシヘモグロビンの光吸収スペクトルはタンパク質の高次構造変化に伴い変化することが過去の研究から知られている。そこで、初期状態をオキシ型で多孔性ガラス中に封入した試料とデオキシ型で封入した試料のデオキシスペクトルを比較してみたところ、両者の差スペクトルは過去に報告されたデオキシRとデオキシTの差スペクトルに大きさ、形が一致した。〈まとめ〉本研究の結果は、2状態モデルの基本仮定の一つとなる4次構造と機能の一対一の関係を支持するとともに、タンパク質の構造と機能の関係を調べる新しい手法としてゾル・ゲル法が使えることを示唆している。
  • 文部科学省:科学研究費補助金(奨励研究(A))
    Date (from‐to) : 1993 -1993 
    Author : 柴山 修哉
     
    1.アロステリックタンパク質共通の分子論的機序を調べる目的で、ヘモグロビン分子の酸素結合における中間状態分子のモデル系をニッケルボルフィリン(デオキシヘムのモデル)による金属置換と分子内架橋(2量体組換え反応の防止)の合併による架橋金属置換混成ヘモグロビンで構築した。今年度行った研究では、全部で8種類ある中間状態分子のうち、研究の重要性が高いと思われる酸素分子の2個結合した4種類の中間状態分子に着目し、これら4分子のモデルとなる高純度試料の調整を行った。2.これらモデルを使った2段階酵素平衝曲線の測定から以下の結果を得た。(1)デオキシヘモグロビンではalphaサブユニットがbetaサブユニットと比較して約3倍高い酵素親和性を示す。(2)酵素化初期段階でのヘム間相互作用の大きさはbeta1beta2、alpha1beta1、alpha1beta2、alpha1alpha2の順に大きくなる。(3)これらのヘム間相互作用はタンパク質の高次構造変化を介して伝わったものと考えてもそれほど大きな矛盾は出てこない。3.本研究の主要な計画の1つに入っていた中間状態分子の高分解能X線結晶解析は予定していたよりも難しい実験であることが解ってきた。顕微分光器によるヘモグロビン結晶の酸素平衝曲線測定で結晶格子力の影響を調べたところ、酸素親和性の高い異常ヘモグロビンを含むほとんどのヒトヘモグロビンは、結晶中でダオキシヘモグロビン型の酸素親和性の低い構造に閉じこめられてしまうことが解った。したがって、中間状態を溶液に近い状態で結晶中に固定することは極めて難しいと思われる。現時点では先ず、結晶格子力のもとになる分子間水素結合に関与するアミノ酸残基を他のアミノ酸に置換した異常ヘモグロビンを大腸菌で合成させ、結晶格子力を減少させたヘモグロビン結晶を作る方向で実験を進めている。
  • 文部科学省:科学研究費補助金(重点領域研究)
    Date (from‐to) : 1992 -1992 
    Author : 西郷 敏, 柴山 修哉
     
    昨年度までの研究で、ミオグロビンの反応中間体のX線吸収スペクトルを急速凍結法により測定し、その構造上の特徴を明かにすることが出来た。本年度は、亜鉛を含むタンパク質分解酵素であるカルボキシペプチダーゼA(CPA)について、その酵素基質複合体を同様の方法で調べた。基質としては、反応の過程を、可視部の蛍光強度の変化で追跡できるダンシル-アラニル-アラニル-フェニルアラニンを用いた。この基質とCPAの反応過程はストップトフロー法により、Auldらのグループにより詳しく調べられ、(1)CPAと基質の混合直後に一つの複合体ES_1が生成し、(2)その後の速い過程によりもう一つの複合体ES_2が生じ、(3)これに続く遅い過程で、基質が分解されることが分かった。反応を遅くし、途中に現れる中間体を十分に蓄積するために、凍結防止剤NaNO_3の共在下、-15℃の低温で反応を観測した。その結果、上に述べたような過程を確認することが出来た。つぎの、反応の開始後、(a)7msと(b)0.6sの時点で急速凍結を行い、得られた試料のZn-k吸収端のX線吸収スペクトルを測定した。(a)と(b)の時点でCPAはそれぞれ主としてES_1とES_2の状態にあると思われる。XANES領域におけるスペクトル(a)は、単独の酵素のスペクトルに良く似ていた。このことはES_1がいわゆるミカエリス複合体であって、基質と亜鉛原子は直接の相互作用をしていないことを意味する。一方、これらに比べ、XANES領域におけるスペクトル(b)は、主ピークが鋭く幅が狭い。金属原子の配位数が4配位から5配位または6配位になるとK吸収端の主ピークが鋭くなるとともに高さが増大することがいくつかの化合物で報告されている。このことから、ES_2の状態では、単独の酵素に比べて、金属まわりの対称性が増加している可能性がある。X線結晶解析の結果に基づいて提案されている反応機構と以上の結果の関連は今後の研究課題である。
  • 文部科学省:科学研究費補助金(重点領域研究)
    Date (from‐to) : 1991 -1991 
    Author : 西郷 敏, 松下 正, 柴山 修哉
     
    高速反応に伴うX線吸収スペクトルの変化を測定するためには、特別の工夫が必要である。一つの方法は、急速凍結により、反応を停止させ、その後に時間をかけてスペクトルを測定することである。本研究は、従来の経験を基礎に、このような目的のための実用的な装置を開発し、いくつかの測定を行った。西洋わさびペルオキシダ-ゼは、反応の過程でいくつかの中間体が出現する。このうち複合体IIのヘム鉄近傍の講造について、X線吸収スペクトル法による二つの異なる結果が報告されている。急速凍結法により複合体IIを調製し、そのX線吸収スペクトルを測定した。その結果、X線の照射により鉄吸収端の位置が低エネルギ-側に移動し、ヘム鉄が還元を受けることが明らかになった。信頼のおけるデ-タを得るためには、このような還元を抑えなければならない。ラジカルと反応性の高いいくつかの物質について調べたところ、ジメチルスルホキシドがX線による還元を大幅に抑えることが分かった。この物質の共存下で複合体IIの測定を行う予定である。ジペプチドやそのアナロダはインヒビタ-としてサ-モライシンに結合するが、分解されない。さまざまなインヒビタ-と酵素の複合体のX線結晶解析がなされ、相互作用の様子が明らかにされている。結晶講造解析のなされている5種類のインヒビタ-とサ-モライシンの複合体の亜鉛吸収端近傍のX線吸収スペクトルを測定した。インヒビタ-のカルボキシル基のO_<ε1>,カルボニル基のO、ホスホリル基のOが水分子のOに代わって配位する場合には、スペクトルの主ピ-クの低エネルギ-側の肩が単独の酵素に比べて顕著に現れた。今後、これらの知見をもとにして、酵素と基質が反応するときに生ずる中間体のスペクトルを測定、解析する予定である。
  • 文部科学省:科学研究費補助金(重点領域研究)
    Date (from‐to) : 1990 -1990 
    Author : 西郷 敏, 松下 正, 柴山 修哉
     
    金属錯体や金属タンパク質などの反応過程をX線吸収スペクトルの時間変化として捉える方法として、分散型測定法と急速凍結法について、装置の開発とその応用を行った。分散型測定では、試料は液体の溶液状態であり、測定中に機械的な動きがないために、スペクトルの微妙な差を検出できるという特徴がある。これまでに我々は、0.3M程度の鉄イオンの溶液反応を時間分解能5msで測定している。測定対象をさらに広げるための重要な条件は、より希薄な試料で質の良いデ-タを取れるようにすることである。このために測定系の改良に着手した。具体的には、X線検出用のフォトダイオ-ドアレイを低暗電流のMOS型のものに変え、これを低雑音の読み出し回路で駆動するようにした。これらを新しいデ-タ処理システムに接続して、動作テストを行った。実際のX線測定は今後行う予定である。希薄な試料のX線吸収スペクトルを測定するためには、蛍光法を利用することが必要になる。急速凍結法を用いれば、このような測定が反応を停止した試料について可能になる。この方法を用いて、シアンを結合した3価鉄ミオグロビンをジチオナイトで急速に還元したときに現れる中間体のX線吸収スペクトルを測定した。鉄吸収端付近の中間体のスペクトルの形は、初期状態のシアン型ものによく似ていて、最終状態の還元型のものとは異なっていた。吸収端の位置は、反応に伴って約4eV低エネルギ-側に移動するが、中間体ではこの値の約1/3だけしか移動していなかった。EXAFS部分をスペクトルから取り出して比較すると、中間体と初期状態は似ているが、最終状態はこれらと大きく異なっていた。以上のことより、中間体へ移行した段階では、シアンはヘム鉄に結合したままで、大きな構造変化は起こっていないことが結論できる。
  • Study on the allosteric mechanism of hemoglobin.
    Date (from‐to) : 1983


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