酵母朊蛋白Sup35NM的寡聚化过程研究  

Oligomerization of yeast prion Sup35NM

作　　者：王艳景 吴奇[1,2] WANG YanJing;WU Chi(Department of Chemistry, The Chinese University ofHong Kong, Hong Kong, China;Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemical Physics, University of Science and Technology of China, Hefei 230026, China)

机构地区：[1]香港中文大学化学系,中国香港 [2]中国科学技术大学化学物理系,合肥微尺度物质科学国家研究中心,合肥230026

出　　处：《中国科学：生命科学》2018年第6期662-670,共9页Scientia Sinica(Vitae)

基　　金：香港特别行政区专项基金(批准号：CUHK4042/13P,2130349,4053060)资助

摘　　要：本文研究了与神经退行性疾病相关蛋白有类似聚集行为的模型蛋白质酵母朊蛋白Sup35NM的错误折叠过程,特别是初始的寡聚化过程.为了延缓和阻止朊蛋白Sup35NM在磷酸盐缓冲液中(体外条件)的聚集以利研究,在Sup35NM的N结构域第31位点处突变并修饰了一个聚合物分子PNIPAM(即蛋白质-高分子结合物Sup35NM-31m-PNIPAM).硫代黄素T荧光光谱实验显示,修饰延缓了Sup35NM的寡聚化,并给出了研究寡聚化的时间窗口:25℃时,修饰前后分别是~10和24 h.激光光散射实验进一步证实了修饰阻碍Sup35NM寡聚化聚集.运用Smoluchowski模型公式,拟合了激光光散射实验数据,定量地给出并对比了修饰前后两个样品在聚集初期(6 h内)的寡聚体分布.结果显示,在引发聚集6 h后,未修饰样品中的蛋白质单体仅剩13%,而在PNIPAM修饰后的样品中蛋白质单体则仍占46%.本工作为可控、定量研究朊蛋白寡聚化提供了新思路.We have studied the misfolding process of a neurodegenerative disease-related protein,yeast prion Sup35NM,especially the initial oligomerization prcoess.To slow down the aggregation rate of Sup35NM in phasphate buffer saline in vitro,we conjugated a commercial available maleimide terminated-poly（N-isopropylacrylamide）（PNIPAM）molecule at the specific 31st residue site on Sup35NM by site-directed mutagenesis and thiol-ene click chemistry,which results in a Sup35NM-31m-PNIPAM conjugate.The aggregation process of Sup35NM was hindered after modification by PNIPAM at 25°C.The Th T fluorescence assay provided a time window for studying the oligomerization prcoess（or lag phase）of Sup35NM（~10 h）and the Sup35NM-31m-PNIPAM conjugate（~24 h）based on the onset of the fluorescence intensity.Further,the results of LLS confirmed the inhibition effect of PNIPAM modification on the oligomerization of Sup35NM due to its high sensitivity on the small aggregates.Finally,the Smoluchowski coagulation equation was adopted to analyze the data from laser light scattering,which provides us quantitative results on the lag phase kinetics and oligomer distribution of the two samples during the initial 6 h upon aggregation.The results show that there only exsits^13%monomers in the sample of the Sup35NM at 6 h after initiating the protein association process,while nearly half of the protein chains exsit as monomers for the Sup35NM-31m-PNIPAM conjugate.In summary,the current work provides a controlable and quantative way to study the oligomerization behavior of the amyloidogenic proteins.

关 键 词：蛋白质折叠 神经退行性疾病 Sup35NM朊蛋白 蛋白质的定点高分子修饰 寡聚化过程 

分 类 号：Q51[生物学—生物化学]