Lys121-Ile的定点突变对人尿胰蛋白酶抑制剂热稳定性的影响

Effect of Mutation from Tyr121 to Ile on the Thermal Stability of UTI

为检验人尿胰蛋白酶抑制剂(UTI)两个结构域中的盐键的稳定性对UTI热变性可逆性的影响,将UTI中的Lys121突变成Ile以中断Glu69与Lys121之间形成的盐键,并命名为?UTI.然后对?UTI进行表达纯化和热解折叠实验.结果显示,?UTI在大肠杆菌中成功表达,纯化复性后经园二色谱测定三级结构与UTI基本相似,酶学测定表明恢复胰蛋白酶抑制剂活性.热变性实验表明:在pH低于7.4的缓冲溶液中?UTI的热解折叠是可逆的,与UTI的结果(pH<4.2)相比,pH值提高了3.结合以前对UTI在酸性条件下可逆热解折叠而在中性与碱性条件下不可逆热解折叠的机制的研究结果,认为多结构域蛋白解折叠的曲线除考虑各个结构域单独形成的多个S曲线的叠加外,还要考虑到结构域之间相互作用强度的影响.

In order to verify the effect of the stability of salt bond on the reversible and irreversible thermal unfolding of Urinary Trypsin Inhibitor（UTI） which was characterized in previous study, the salts band between Glu69 and Lys121 in UTI was ruptured by mutating Tyr121 to Ile, which was name as △UTI. It could be induced that the thermal unfolding of △ UTI should be reversible in a higher pH if previous induction was correct. And then △ UTI was expressed, purified and characterized the experiments of thermal unfolding of △ UTI were preformed too. The results showed： △ UTI can be successively expressed; the tertiary structure of △ UTI was similar to the one of native UTI and △ UTI had the same inhibitor activity as UTI, the thermal unfolding of △ UTI was still reversible when pH was lower than 7.4, which was 3 pH high compared with previous result. The result was consistent with the induction. Combined with results of other＇s scientists, we believed that the effect of interacting intensity among different domains on the curves of unfolding should be taken into account during unfolding of multi-domain proteins.