1997 年度実績報告書

タンパク質の構造安定化機構への温度・圧力効果の研究

研究課題

研究課題/領域番号	08045025
研究機関	立命館大学
研究代表者	澤村精治立命館大学, 理工学部, 助教授 (10167439)
研究分担者	ペィティグレンフェルブリティッシュコロンビア大学, 理学部, 教授マクギリブレイロスブリティッシュコロンビア大学, 理学部, 教授マウクグラントブリティッシュコロンビア大学, 医学部, 教授加藤稔立命館大学, 理工学部, 講師 (00241258) 谷口吉弘立命館大学, 理工学部, 教授 (70066702)
キーワード	タンパク質 / 高圧力 / リゾチーム
研究概要	最初にタンパク質の存在する水中での溶質溶媒相互作用についても調べた。そのいくつかを示す。タンパク質の疎水部分のモデルとして四級アンモニウム塩を用い、この水溶液のNMR緩和の回転アニソトロピーから3種の水和水の存在を確認した。リゾチーム-アルコール-水系の熱測定からは、タンパク質表面の結合サイトのアルコールによる占有はそのアルコールにとって必ずしもタンパク質の性質に影響を及ぼさなくてもよいことを明らかにした。リゾチームの水への高圧溶解度を測定した。一方でタンパク質の構成部分であるアミノ酸の中でもっとも単純なグリシンについても高圧溶解度を測定した。その結果、リゾチームの溶解度は加圧とともに増加するのに対し、グリシンは減少した。両者とも加熱すると溶解度は増加することから、溶解度の圧力依存性を調べることの方が、タンパク質と水との相互作用についての情報が得られやすいことが示唆された。タンパク質については、クジラミオグロビン(Mb)のFT-IRスペクトルを数千気圧の高圧下で-10〜70℃で測定した。その結果、圧力によるMbの二次構造は二状態転移を示し、加熱によりMbの二次構造は分子間ベータシートの形成を伴いながらランダムコイルへと不可逆的に転移すること、さらに低温ではMbの二次構造はランダムコイルへと可逆的に変化する事を明らかにした。また、遺伝子工学的手法によるタンパク質試料の調製を試みた。その結果、大腸菌を利用する事により酵母チトクロムCを調整することが可能となり、ワイルドタイプのチトクロムCが比較的高収率で得られることとなった。このことによりさらに異なったタイプのチトクロムCも調製できる可能性を示し、耐熱・耐圧を有するタンパク質を調製する道が開けた。

研究成果
(33件)

すべてその他

すべて文献書誌 (33件)

[文献書誌] T.Nakai: "Effects of Pressure and Temperatur on the viscosity 13 coefficiant for Potassium Chloride in Water" Matter.Sci.Res.Int.,. 3. 204-209 (1997)
[文献書誌] N.Takeda: "Pressure-induced Structural rearrangements of bovine pancreatic trypsin inhibitor studied by FTIR Spectroscopy" Biospectroscopy. 印刷中. (1998)
[文献書誌] T.Matsumoto: "Effect of Pressure on the mechanism of hydrolysis of malcternose,maltopcrvtaose,and maltohexose catalyzed by porcine paticieatic α-amylase" Biochim.Biophys.Acta.1343. 243-250 (1997)
[文献書誌] K.Fumino: "NMR Studies on dynamic structure of hydrated water molecules in tetraalkylammonium bromide-D_2O Solutions at5-25℃" J.Mol.Liquids,. 72、(印刷中).
[文献書誌] K.Fumino: "Concontration dependence of nuclear magnetic relaxation vntes of alkalimetal ionic nucliein dilute solutions" Denki Kagaku. 65. 198-203 (1997)
[文献書誌] Y.Koga: ""Intermolecular Interactions"in Aqueous Solutions of Three Components including Lysozyme" Fluid Phase Egnil.(印刷中). (1998)
[文献書誌] I.J.F.Day: "Ion Solration in Binavy Mixtares" J.Chem.Phys.106. 2782-2793 (1997)
[文献書誌] G.Ayton: "Liquid Crystal Phases of Dipolar Discotic Particles" Phys.Rer.E. 55. 447-454 (1997)
[文献書誌] M.Twitchett: "Intramolecular Electron Transfer Kinetics of a Synthetin Flavocytochrome C" J.Am.Chem.Soc.119. 435-436 (1997)
[文献書誌] J.Bujons: "Charge Reversal of a Critical Active Site Residue of Cytochrome C Peroxidase:Cfaracterixation of the Afs 48Glu Variant" Eur.J.Biochem. 243. 72-84 (1997)
[文献書誌] C.L.Hunter: "Role of Heme Propionates in the Interaction of Heme with Apomyoglobin and Apocytochrome b5." Biochemistry. 36. 1010-1017 (1997)
[文献書誌] C.L.Hunter: "Dissociation of Heme from Myoglobin and Cytochrome b5:Comparison of Behavior in Solution and the Gas Phase" Biochemistry. 36. 1018-1025 (1997)
[文献書誌] J.M.Nocek: "Cyclic Electron Transfer with in the [Zn-Deuteremyoglobin,Cytochromb5]Complex:Redox-Linked Changes at theInterface" J.Am.Chem.Lec.119. 2146-2155 (1997)
[文献書誌] A.G.Mank: "Control of Metalloprotein Redox Potentials:What Does Site-Directed Mutagenesis of Hemoproteins Tell Us ?" J.Biol.Inors.Chem.2. 119-125 (1997)
[文献書誌] K.Kishi: "Site-Directed Mutations of Phonylalanire-190 of Manganese Peroxidase:Effects on Stability,Function and Coordination" Biochemistry. 36. 4268-4277 (1997)
[文献書誌] L.Konermann: "Acid-Induced Donaturation of Myoglobin Studied by Time-Resolved Electrospray Ionization Mass Spectrometry" Biochemistry. 36. 6448-6454 (1997)
[文献書誌] R.Maurus: "A Myoglobin Variant witha Polar Substitution in Conserved Hydrophobic Cluster in the Heme Binding Pocket" Biochim.Biophys.Acta.1341. 1-13 (1997)
[文献書誌] Cote.H.C.F: "A Novel approach for the characterization and epitope determination of alupus anticoagnlant-associatedneutralizing anti-prothrombin antibody" Am.J.Clin.Phthol.107. 197-205 (1997)
[文献書誌] P.Westh: "Intermolecular Interactions of Lysozyme and Small Alcohols:A Calorimetric Investigation" J.Phys.Chem. B101. 5755-5758 (1997)
[文献書誌] Y.Xu: "Light Scattering Study of Sol-Gel Trsnsformation of Sio_<> Colloid at Extremely High Concentrations" Clays and Clay Minerals. (印刷中). (1998)
[文献書誌] D.R.Berard: "Structive of the Metal-Aqueous Electrolyte Solution Interface" J.Chem.Phys.107. 4719-4728 (1997)
[文献書誌] J.C.Shelley: "Modeling and Structure of Mercury-Water Interface" J.Chem.Phys.107. 2122-2141 (1997)
[文献書誌] G.Ayton: "Ferroelectic and Dipolar Glass Phases of Non-Crystalline Systems" Phys.Rev.E. 56. 562-570 (1997)
[文献書誌] N.M.Cann: "An Investigation of the Infulence of Solute Size and Insertion on Solvation Thermodynamics" J.Chem.Phys.106. 8165-8195 (1997)
[文献書誌] Cote.H.C.F.: "Functional Characterization of recombinant human meizo-thrombin and meizothrombin(DesF1)" J.Biol.Chem.272. 6194-6200 (1997)
[文献書誌] M.A.Brown: "Vectors for expressing proteinsat the amino-terminus of an activationdomain for use in the yeast two-hybridsystem" Anal.Biochem.247. 451-452 (1997)
[文献書誌] Q.Y.He: "Effects of mutations of aspartic acid63 on the metal-binding properties of the vecanbinant N-lobe of humanserum transferrin" Biochemistry. 36. 5522-5528 (1997)
[文献書誌] L.A.Luck: "19F NMR Studies of the recombinant human transferrin N-lobe and three singlepoint mutants" Mag.Reson.Chem.35. 477-481 (1997)
[文献書誌] A.B.Mason: "Receptor recognition Sites reside in both lobes of Humann Serum transferrin" Biochem.J.326. 77-85 (1997)
[文献書誌] O.Zak: "A kinetically active site in the C-lobe of human transferrin" Biochemistry. 36. 11036-11043 (1997)
[文献書誌] M.M.Guarna: "On-line monitoring and control of methanol concentration in Shake-flask cultures of Pichia pastoris" Biotechnol.Bioerg.56. 279-286 (1997)
[文献書誌] Q-Y.He: "Inequivalence of the two tyrosine ligandsin the N-lobe of human serumtransferrin" Biochemistry. 36. 14853-14860 (1997)
[文献書誌] 谷口吉弘: "生物・環境産学のための非加熱プロセス事典" サイエンスフォーラム, 95-99 (1997)

1997 年度 実績報告書

タンパク質の構造安定化機構への温度・圧力効果の研究

研究代表者

澤村 精治 立命館大学, 理工学部, 助教授 (10167439)

研究成果

[文献書誌] T.Nakai: "Effects of Pressure and Temperatur on the viscosity 13 coefficiant for Potassium Chloride in Water" Matter.Sci.Res.Int.,. 3. 204-209 (1997)

[文献書誌] N.Takeda: "Pressure-induced Structural rearrangements of bovine pancreatic trypsin inhibitor studied by FTIR Spectroscopy" Biospectroscopy. 印刷中. (1998)

[文献書誌] T.Matsumoto: "Effect of Pressure on the mechanism of hydrolysis of malcternose,maltopcrvtaose,and maltohexose catalyzed by porcine paticieatic α-amylase" Biochim.Biophys.Acta.1343. 243-250 (1997)

[文献書誌] K.Fumino: "NMR Studies on dynamic structure of hydrated water molecules in tetraalkylammonium bromide-D_2O Solutions at5-25℃" J.Mol.Liquids,. 72、(印刷中).

[文献書誌] K.Fumino: "Concontration dependence of nuclear magnetic relaxation vntes of alkalimetal ionic nucliein dilute solutions" Denki Kagaku. 65. 198-203 (1997)

[文献書誌] Y.Koga: ""Intermolecular Interactions"in Aqueous Solutions of Three Components including Lysozyme" Fluid Phase Egnil.(印刷中). (1998)

[文献書誌] I.J.F.Day: "Ion Solration in Binavy Mixtares" J.Chem.Phys.106. 2782-2793 (1997)

[文献書誌] G.Ayton: "Liquid Crystal Phases of Dipolar Discotic Particles" Phys.Rer.E. 55. 447-454 (1997)

[文献書誌] M.Twitchett: "Intramolecular Electron Transfer Kinetics of a Synthetin Flavocytochrome C" J.Am.Chem.Soc.119. 435-436 (1997)

[文献書誌] J.Bujons: "Charge Reversal of a Critical Active Site Residue of Cytochrome C Peroxidase:Cfaracterixation of the Afs 48Glu Variant" Eur.J.Biochem. 243. 72-84 (1997)

[文献書誌] C.L.Hunter: "Role of Heme Propionates in the Interaction of Heme with Apomyoglobin and Apocytochrome b5." Biochemistry. 36. 1010-1017 (1997)

[文献書誌] C.L.Hunter: "Dissociation of Heme from Myoglobin and Cytochrome b5:Comparison of Behavior in Solution and the Gas Phase" Biochemistry. 36. 1018-1025 (1997)

[文献書誌] J.M.Nocek: "Cyclic Electron Transfer with in the [Zn-Deuteremyoglobin,Cytochromb5]Complex:Redox-Linked Changes at theInterface" J.Am.Chem.Lec.119. 2146-2155 (1997)

[文献書誌] A.G.Mank: "Control of Metalloprotein Redox Potentials:What Does Site-Directed Mutagenesis of Hemoproteins Tell Us ?" J.Biol.Inors.Chem.2. 119-125 (1997)

[文献書誌] K.Kishi: "Site-Directed Mutations of Phonylalanire-190 of Manganese Peroxidase:Effects on Stability,Function and Coordination" Biochemistry. 36. 4268-4277 (1997)

[文献書誌] L.Konermann: "Acid-Induced Donaturation of Myoglobin Studied by Time-Resolved Electrospray Ionization Mass Spectrometry" Biochemistry. 36. 6448-6454 (1997)

[文献書誌] R.Maurus: "A Myoglobin Variant witha Polar Substitution in Conserved Hydrophobic Cluster in the Heme Binding Pocket" Biochim.Biophys.Acta.1341. 1-13 (1997)

[文献書誌] Cote.H.C.F: "A Novel approach for the characterization and epitope determination of alupus anticoagnlant-associatedneutralizing anti-prothrombin antibody" Am.J.Clin.Phthol.107. 197-205 (1997)

[文献書誌] P.Westh: "Intermolecular Interactions of Lysozyme and Small Alcohols:A Calorimetric Investigation" J.Phys.Chem. B101. 5755-5758 (1997)

[文献書誌] Y.Xu: "Light Scattering Study of Sol-Gel Trsnsformation of Sio_<> Colloid at Extremely High Concentrations" Clays and Clay Minerals. (印刷中). (1998)

[文献書誌] D.R.Berard: "Structive of the Metal-Aqueous Electrolyte Solution Interface" J.Chem.Phys.107. 4719-4728 (1997)

[文献書誌] J.C.Shelley: "Modeling and Structure of Mercury-Water Interface" J.Chem.Phys.107. 2122-2141 (1997)

[文献書誌] G.Ayton: "Ferroelectic and Dipolar Glass Phases of Non-Crystalline Systems" Phys.Rev.E. 56. 562-570 (1997)

[文献書誌] N.M.Cann: "An Investigation of the Infulence of Solute Size and Insertion on Solvation Thermodynamics" J.Chem.Phys.106. 8165-8195 (1997)

[文献書誌] Cote.H.C.F.: "Functional Characterization of recombinant human meizo-thrombin and meizothrombin(DesF1)" J.Biol.Chem.272. 6194-6200 (1997)

[文献書誌] M.A.Brown: "Vectors for expressing proteinsat the amino-terminus of an activationdomain for use in the yeast two-hybridsystem" Anal.Biochem.247. 451-452 (1997)

[文献書誌] Q.Y.He: "Effects of mutations of aspartic acid63 on the metal-binding properties of the vecanbinant N-lobe of humanserum transferrin" Biochemistry. 36. 5522-5528 (1997)

[文献書誌] L.A.Luck: "19F NMR Studies of the recombinant human transferrin N-lobe and three singlepoint mutants" Mag.Reson.Chem.35. 477-481 (1997)

[文献書誌] A.B.Mason: "Receptor recognition Sites reside in both lobes of Humann Serum transferrin" Biochem.J.326. 77-85 (1997)

[文献書誌] O.Zak: "A kinetically active site in the C-lobe of human transferrin" Biochemistry. 36. 11036-11043 (1997)

[文献書誌] M.M.Guarna: "On-line monitoring and control of methanol concentration in Shake-flask cultures of Pichia pastoris" Biotechnol.Bioerg.56. 279-286 (1997)

[文献書誌] Q-Y.He: "Inequivalence of the two tyrosine ligandsin the N-lobe of human serumtransferrin" Biochemistry. 36. 14853-14860 (1997)

[文献書誌] 谷口 吉弘: "生物・環境産学のための非加熱プロセス事典" サイエンスフォーラム, 95-99 (1997)

1997 年度実績報告書

澤村精治立命館大学, 理工学部, 助教授 (10167439)

[文献書誌] 谷口吉弘: "生物・環境産学のための非加熱プロセス事典" サイエンスフォーラム, 95-99 (1997)