Thermostabilization mechanism of proteins from thermophiles

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。

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.