Takano K, Yamagata Y, Yutani K
Institute for Protein Research, Osaka University, Yamadaoka, Suita, 565-0871, Japan.
J Mol Biol. 1998 Jul 24;280(4):749-61. doi: 10.1006/jmbi.1998.1906.
To get a general rule for the relationship between hydrophobic effect and conformational stability, five Ile to Val and nine Val to Ala mutants (3SS mutants) from 3SS (C77A/C95A) human lysozyme were constructed. As known from previous studies, the 3SS protein lacking a disulfide bond between Cys77 and Cys95 is destabilized by enthalpic factors, as revealed by a decrease of about 20 kJ/mol in the denaturation Gibbs energy change (DeltaG) value, as compared to the wild-type protein, which has four disulfide bonds. In this study, the stabilities and structures of the 3SS mutants were determined by differential scanning calorimetry and X-ray crystal analysis, respectively, and compared with those of the mutants (4SS mutants) from the wild-type (4SS) protein published previously. The stabilities of all the 3SS mutants, except for V110A-3SS were decreased as compared with that of the 3SS protein, coinciding with the results for the 4SS mutants. The change in the denaturation Gibbs energy change (DeltaDeltaG) values of the 3SS mutants relative to the 3SS protein at the denaturation temperature (49.2 degreesC) of the 3SS protein at pH 2.7 were similar to those of the equivalent 4SS mutants relative to the wild-type at 64.9 degreesC. The Delta DeltaG values of the 3SS mutants correlated with the changes in hydrophobic surface area exposed upon denaturation (Delta DeltaASAHP) for all of the hydrophobic residues when the effects of the secondary structure propensity were considered. This correlation is identical with that previously found for the 4SS mutants. The linear relation between Delta DeltaG and Delta DeltaASAHP for all of the hydrophobic residues with the same slope was found also for the mutants of T4 lysozyme already reported, indicating that this is a general relationship between changes in conformational stability and changes in ASA values of hydrophobic residues due to mutations.
为了得出疏水效应与构象稳定性之间关系的一般规律，构建了来自3SS（C77A/C95A）人溶菌酶的5个异亮氨酸突变为缬氨酸和9个缬氨酸突变为丙氨酸的突变体（3SS突变体）。如先前研究所知，与具有四个二硫键的野生型蛋白相比，在77位半胱氨酸和95位半胱氨酸之间缺少二硫键的3SS蛋白因焓因素而不稳定，变性吉布斯自由能变化（ΔG）值降低约20 kJ/mol。在本研究中，分别通过差示扫描量热法和X射线晶体分析确定了3SS突变体的稳定性和结构，并与先前发表的野生型（4SS）蛋白的突变体（4SS突变体）进行了比较。与3SS蛋白相比，除V110A-3SS外，所有3SS突变体的稳定性均降低，这与4SS突变体的结果一致。在pH 2.7时，3SS蛋白变性温度（49.2℃）下，3SS突变体相对于3SS蛋白的变性吉布斯自由能变化（ΔΔG）值，与等效4SS突变体在64.9℃相对于野生型的变化相似。当考虑二级结构倾向的影响时，3SS突变体的ΔΔG值与所有疏水残基变性时暴露的疏水表面积变化（ΔΔASAHP）相关。这种相关性与先前在4SS突变体中发现的相同。对于已报道的T4溶菌酶突变体，也发现了所有疏水残基的ΔΔG与ΔΔASAHP之间具有相同斜率的线性关系，这表明这是构象稳定性变化与突变导致的疏水残基ASA值变化之间的一般关系。
