Takano K, Ogasahara K, Kaneda H, Yamagata Y, Fujii S, Kanaya E, Kikuchi M, Oobatake M, Yutani K
Institute for Protein Research, Osaka University, Japan.
J Mol Biol. 1995 Nov 17;254(1):62-76. doi: 10.1006/jmbi.1995.0599.
In order to understand the contribution of hydrophobic residues to the conformational stability of human lysozyme, five Ile mutants (Ile --> Val) in the interior of the protein were constructed. The thermodynamic parameters characterizing the denaturation of these mutant proteins were determined by scanning calorimetry, and the three-dimensional structure of each mutant protein was solved at high resolution by X-ray crystallography. The thermodynamic analyses at 64.9 degrees C and at pH 2.7 revealed the following. (1) The stabilities of all the mutant proteins were decreased as compared with that of the wild-type protein. (2) The changes in the calorimetric enthalpies were larger than those in the Gibbs energies, and were compensated by entropy changes. (3) The destabilization mechanism of the mutant proteins differs, depending on the location of the mutation sites. X-ray analyses showed that the overall structures of all the mutant human lysozymes examined were identical to that of the wild-type protein, and only small structural rearrangements were observed locally around some of the mutation sites. The most striking change among the mutant proteins was found in the mutant protein, 159V, which contains a new water molecule in the cavity created by the mutation. The thermodynamic stabilities of the mutant proteins are discussed in light of the high-resolution X-ray structures of the wild-type and five mutant human lysozymes examined.
为了了解疏水残基对人溶菌酶构象稳定性的贡献，构建了该蛋白质内部的五个异亮氨酸突变体（异亮氨酸→缬氨酸）。通过扫描量热法测定了表征这些突变蛋白变性的热力学参数，并通过X射线晶体学高分辨率解析了每个突变蛋白的三维结构。在64.9℃和pH 2.7条件下的热力学分析结果如下：（1）与野生型蛋白相比，所有突变蛋白的稳定性均降低。（2）量热焓变大于吉布斯自由能变，并由熵变补偿。（3）突变蛋白的去稳定机制因突变位点的位置而异。X射线分析表明，所有检测的突变型人溶菌酶的整体结构与野生型蛋白相同，仅在一些突变位点周围局部观察到小的结构重排。在突变蛋白159V中发现了最显著的变化，该突变蛋白在突变产生的腔内含有一个新的水分子。根据所检测的野生型和五个突变型人溶菌酶的高分辨率X射线结构，讨论了突变蛋白的热力学稳定性。
