Thermostabilization mechanism of proteins from thermophiles

嗜热菌蛋白质的热稳定机制

• 批准号: 04044109
• 负责人: YUTANI Katsuhide
• 金额: $ 4.29万
• 依托单位: Institute for Protein Resaerch, Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04044109/
• 关键词: Calorimetry; Thermostability; Protein denaturation; Thermodynamics; Thermophile; 疎水性相互作用; 好熱菌; トリプトファン合成酵素; 好熱菌酵素; 耐熱性蛋白質; カロリメーター; 蛋白質の安定性; トリプトファンシンターゼ; 熱力学的パラメーター; 変性のエンタルピー; 変性のエントロピー

Studies on extremely thermostable proteins that have denaturation temperature of more than 100 ^OC help us to understand the stabilization mechanism of a general globule protein, since thermodynamic parameters of the unfolding(such as enthalpy, entropy, and heat capacity changes)are assumed to have the special characteristics close to 110 ^OC(for example, a common converged value), which should be confirmed by actual measurements of the parameters at that temperature using extremely thermostable proteins. Each of genes cording sequence of alpha-amylase, pyroglutamine peptidase (PGP), and methionine aminopeptidase (MAP)from superthermophile, Pyrococcus furiosus was inserted in an expression vector for Escherichia coli. The three proteins expressed in a mesophile had properties similar to those observed in the native proteins. The denaturation temperature of each purified protein was found to be more than 100 ^OC by calorimetry. Thermodynamic parameters of unfolding for the three extremely thermostable proteins were obtained using a scanning calorimeter, DASM4 at various pHs. Their values did not coincide with those estimated from methophilic proteins, suggesting that there are some problems for the extrapolation to 110 ^OC.PGP was found to be a tetramer protein consisting of two dimmers with an inter-molecular disulfide bond(due to Cys 118). From comparison of calorimetric results of the wild-type PGP with those of the mutant protein(C118S)substituted by Ser at Cys118, we found that the wild-type protein was greatly stabilized by the intermolecular disulfide bond, although a disulfide bond which might be broken in high temperature has never seen in thermophilic proteins. On the other hands, kinetic studies of unfolding and refolding of PGP indicated that thermostabilization of the protein is caused by-extremely lowering the unfolding rate.

对变性温度超过100 ℃的极端热稳定蛋白质的研究有助于我们理解一般球状蛋白质的稳定机制，因为去折叠的热力学参数（如焓，熵和热容变化）被假定为具有接近110 ^OC的特殊特性（例如，一个共同的收敛值），这应该通过在该温度下使用极端热稳定的蛋白质对参数的实际测量来确认。将来自超嗜热菌激烈火球菌的α-淀粉酶、焦谷氨酰胺肽酶（PGP）和甲硫氨酸氨基肽酶（MAP）的基因序列分别插入大肠杆菌的表达载体中。在嗜温细胞中表达的三种蛋白质具有与在天然蛋白质中观察到的那些相似的性质。通过量热法发现每个纯化蛋白的变性温度大于100 ° C。使用扫描量热仪DASM 4在不同pH值下获得了三种极端热稳定蛋白质的展开热力学参数。它们的值与嗜热蛋白的估计值不一致，表明外推到110 ^OC存在一些问题。发现PGP是由两个二聚体组成的四聚体蛋白，分子间有一个二硫键（由于Cys 118）。通过比较野生型PGP与Cys 118位被Ser取代的突变蛋白（C118 S）的量热结果，我们发现野生型蛋白被分子间二硫键极大地稳定，尽管在高温下可能断裂的二硫键在嗜热蛋白中从未见过。另一方面，PGP的去折叠和重折叠动力学研究表明，蛋白质的热稳定性是由于极大地降低去折叠速率。

项目成果

Ryota KUROKI, Katsutoshi NITTA, and Katsuhide YUTANI: "Thermodynamic Changes in the Binding of Ca to a Mutant Human Lysozyme (D86/92): Enthalpy-enthropy Compensation Obseved upon Ca Binding to Proteins" J. Biol. Chem.267. 24297-24301 (1992)

Ryota KUROKI、Katsutoshi NITTA 和 Katsuhide YUTANI：“Ca 与突变人溶菌酶 (D86/92) 结合的热力学变化：Ca 与蛋白质结合时观察到的熵-熵补偿”J. Biol。

K.Yutani: "Conformational stability of a protein" PNE,Protein, Nuclecic acid and Enzyme. 39. 1017-1027 (1994)

K.Yutani：“蛋白质的构象稳定性”PNE、蛋白质、核酸和酶。

M.Odaka,et al.: "Parrael effects of Tyr-341 mutations of the TF1-ATPase β subunit on the Kd of the Isolated β subunit" J.Biochem.(in press). (1994)

M.Odaka 等人：“TF1-ATPase β 亚基的 Tyr-341 突变对分离的 β 亚基的 Kd 的平行影响”J.Biochem.（出版中）。

M.Odaka,C.Kaibara,T.Amano,T.Matsui,E.Muneyuki,K.Ogasahara,K.Yutani,& M.Yoshida: "Tyr-341 of the β subunit is a major Km-determining redsidue of TF1-ATPase:Parallel effect of its mutations on Kd(ATP) of the β subunit and on Km(ATP) of the α3β3γ complex." J

M. Odaka、C. Kaibara、T. Amano、T. Matsui、E. Muneyuki、K. Ogasahara、K. Yutani 和 M. Yoshida：“β 亚基的 Tyr-341 是 TF1 的主要 Km 决定残基-ATP酶：其突变对β亚基的Kd(ATP)和α3β3γ复合物的Km(ATP)的平行影响。" J

K.Ogasahara & K.Yutani: "Unfolding-refolding kinetics of the tryptophan synthase α subunit by the CD and fluorescence measurements." J.Mol.Biol.235(in press). (1994)

K.Ogasahara 和 K.Yutani：“CD 和荧光测量的色氨酸合酶 α 亚基的解折叠-重折叠动力学。”J.Mol.Biol.235（出版中）。