Perry L J, Wetzel R
Biochemistry. 1986 Feb 11;25(3):733-9. doi: 10.1021/bi00351a034.
We have introduced an intramolecular disulfide bond into T4 lysozyme and have shown this molecule to be significantly more stable than the wild-type molecule to irreversible thermal inactivation [Perry, L.J., & Wetzel, R. (1984) Science (Washington, D.C.) 226, 555-557]. Wild-type T4 lysozyme contains two free cysteines, at positions 54 and 97, and no disulfide bonds. By directed mutagenesis of the cloned T4 lysozyme gene, we replaced Ile-3 with Cys. Oxidation in vitro generated an intramolecular disulfide bond; proteolytic mapping showed this bond to connect Cys-3 to Cys-97. While this molecule exhibited substantially more stability against thermal inactivation than wild type, its stability was further enhanced by additional modification with thiol-specific reagents. This and other evidence suggest that at basic pH and elevated temperatures Cys-54 is involved in intermolecular thiol/disulfide interchange with the engineered disulfide, leading to inactive oligomers. Mutagenic replacement of Cys-54 with Thr or Val in the disulfide-cross-linked variant generated lysozymes exhibiting greatly enhanced stability toward irreversible thermal inactivation.
我们已将分子内二硫键引入T4溶菌酶，并已证明该分子比野生型分子对不可逆热失活具有显著更高的稳定性[佩里，L.J.，& 韦策尔，R.（1984年）《科学》（华盛顿特区）226, 555 - 557]。野生型T4溶菌酶在第54和97位含有两个游离半胱氨酸，且不存在二硫键。通过对克隆的T4溶菌酶基因进行定向诱变，我们将异亮氨酸 - 3替换为半胱氨酸。体外氧化产生了一个分子内二硫键；蛋白酶解图谱显示该键将半胱氨酸 - 3与半胱氨酸 - 97相连。虽然该分子对热失活表现出比野生型显著更高的稳定性，但其稳定性通过用硫醇特异性试剂进行额外修饰进一步增强。这一证据及其他证据表明，在碱性pH和升高温度下，半胱氨酸 - 54参与了与工程化二硫键的分子间硫醇/二硫键交换，导致形成无活性的寡聚体。在二硫键交联变体中用苏氨酸或缬氨酸对半胱氨酸 - 54进行诱变替换，产生了对不可逆热失活表现出极大增强稳定性的溶菌酶。
