Investigation for transition state of loop insertion in serpin : An approach toward prevention of amyloidosis

在某些组织的遗传性分子异常的情况下，丝氨酸蛋白酶抑制物(丝氨酸蛋白酶抑制剂)的构象变化、环插入可能会导致蛋白质凝集。为了控制环路的插入，本项目研究了一种假定的过渡态。卵清蛋白是一种属于丝氨酸蛋白超家族的蛋白，虽然它对丝氨酸蛋白酶没有任何抑制活性，但已成为研究的重点。使用一些卵清蛋白突变体，试图改进环插入的定量测定。以前，卵清蛋白突变体的环插入是通过测量枯草杆菌酶降解有限蛋白的时间过程来观察到的。由于这一过程的复杂性和误差，人们一直渴望找到一些定量和方便的方法。由于离子交换柱层析可以通过蛇纹环的插入来区分构象异构体，我们建立了一种新的定量分析卵清蛋白突变体中的环插入的方法。利用卵清蛋白突变体R339T/A352R，其P1-P1‘位点可与胰酶反应，该新的高效液相色谱方法已被证明是一种简单而准确的方法。由于丝氨酸的结构和功能状况，增加环插入率应该会导致抑制活性的获得。因此，我们在晶体结构数据的基础上进行了进一步的诱变，以加快环的插入速度。另外的突变体K290T/R339T/A352R和R104A/R339T/A352R以及R339T/A352R的二硫键还原形式R339T/A352R的插入率分别是R339T/A352R对照的1.5、3.7和6.7倍。突变和二硫键还原应使远端折叠A结构具有更灵活的性质。

A conformational change, loop-insertion of serine proteinase inhibitors (serpin) may cause protein coagulation in certain tissues in the cases of their inherited molecular-abnormalities. In order to control the loop-insertion, a supposed transition state has been investigated in the present project. Ovalbumin, the albumen protein, which belongs to a superfamily of serpin has been focused although it does not show any inhibitory activity to serine proteinases. Using some ovalbumin mutants, improvement in quantitative determination of loop-insertion has been attempted. Previously, loop-insertion of the ovalbumin mutant had been observed by measuring time-courses of limited proteolysis with subtilisin. Since the procedure had been complicated and erroneous, some quantitative and convenient methods have been longed. As the ion-exchanger column chromatography on HPLC can differentiate the conformational isomer through the serpin loop-insertion, we set up a novel procedure for quantitative analysis of loop-insertion in ovalbumin mutants in the present project. Utilizing an ovalbumin mutant R339T/A352R in which the P1-P1' site is accessible against trypsin, the novel HPLC procedure has been proved to be a simple and accurate procedure. Because of the structural and functional situations of serpin, increased loop insertion rate should lead to the acquisition of the inhibitory activity. We therefore did further mutagenesis to accelerate the loop insertion rate on the basis of the data of crystal structure. Additional mutants, K290T/R339T/A352R and R104A/R339T/A352R, and the disulfide-reduced form of R339T/A352R, respectively, displayed 1.5, 3.7, and 6.7-fold increase in the loop insertion rate as compared with R339T/A352R control. The mutation and disulfide reduction should give a more flexible nature on the distal sheet A structure.