Inaka K, Taniyama Y, Kikuchi M, Morikawa K, Matsushima M
Protein Engineering Research Institute, Osaka, Japan.
J Biol Chem. 1991 Jul 5;266(19):12599-603.
The three-dimensional structure of a mutant human lysozyme, C77/95A, in which residues Cys77 and Cys95 were replaced by alanine, was determined at 1.8-A resolution by x-ray crystallography. The properties of this mutant protein have been well characterized with respect to its thermal stability and secretion efficiency in a yeast expression system. The overall three-dimensional structure of C77/95A was found to be essentially identical to that of the wild-type human lysozyme, although the coordinates were shifted by more than 0.5 A and the thermal factors of the main-chain atoms were increased in the vicinity of residue 77. The reduction in thermal stability of this mutant has been previously explained by an increase in entropy of the unfolded state. In addition, a packing defect (cavity) produced by the removal of the disulfide bond was detected in the three-dimensional structure of C77/95A. This cavity can also be a reason why the stability of the protein is reduced because the free energy of the folded state could be expected to increase. The increased secretion efficiency cannot be due mainly to the three-dimensional structure, but may possibly be related to some event in the pathway of protein secretion. One of the possibilities might involve molecular flexibilities in the secondary or tertiary structure for lack of one of the disulfide bonds.
通过X射线晶体学在1.8埃分辨率下测定了突变型人溶菌酶C77/95A的三维结构，其中半胱氨酸77和半胱氨酸95被丙氨酸取代。该突变蛋白的特性在酵母表达系统中已就其热稳定性和分泌效率进行了充分表征。发现C77/95A的整体三维结构与野生型人溶菌酶基本相同，尽管坐标移动了超过0.5埃，并且在残基77附近主链原子的热因子增加。该突变体热稳定性的降低先前已通过未折叠状态熵的增加来解释。此外，在C77/95A的三维结构中检测到由于去除二硫键而产生的堆积缺陷（腔）。这个腔也可能是蛋白质稳定性降低的原因，因为折叠状态的自由能预计会增加。分泌效率的提高主要不是由于三维结构，而是可能与蛋白质分泌途径中的某些事件有关。其中一种可能性可能涉及由于缺少一个二硫键而导致的二级或三级结构中的分子柔韧性。
