小分子热休克蛋白Mj HSP16.5的分级变性  

Hierarchical Unfolding of Mj HSP16.5

作　　者：王铮[1] 赖兵[1] 曹洁[1] 李竹[1] 瞿丽丽[1] 曹傲能[1,2] 来鲁华[1] 

机构地区：[1]北京大学化学与分子工程学院,北京分子科学国家实验室,分子动态与稳态结构国家重点实验室,北京100871 [2]上海大学纳米化学与生物学研究所,上海200444

出　　处：《物理化学学报》2008年第10期1745-1750,共6页Acta Physico-Chimica Sinica

基　　金：国家自然科学基金(20203001,20673003,30490245)资助项目

摘　　要：应用荧光光谱、圆二色光谱、体积排阻色谱、激光动态光散射等技术,研究了来自嗜热古细菌Methanococcus jannaschii(Mj)的小分子热休克蛋白Mj HSP16.5在变性剂作用下的变性过程.研究表明,在pH7时,Mj HSP16.5在8mol·L-1尿素作用下不会发生变性.在pH7条件下,盐酸胍对Mj HSP16.5的变性表现为一个分级过程,分别在2.0、3.0和6.0mol·L-1盐酸胍浓度附近,出现明显的结构变化;到7.0mol·L-1盐酸胍时,Mj HSP16.5才完全变性.降低溶液pH值将使Mj HSP16.5的变性变得更为容易.Mj HSP16.5 is a small heat shock protein （sHSP） from the hyperthermophilic methanoarchaeon, Methanococcus jannaschii （Mj）, which lives at the environment of high temperature up to 94 ℃. The structural data showed that Mj HSP16.5 was a 24-mer that formed a hollow sphere with octahedral symmetry. Mj HSP16.5 was very stable at pH 7 that it maintained the 24-mer structure even at 85 ℃. In the present study, we investigated the unfolding process of Mj HSP16.5 in the presence of denaturants using several techniques, including circular dichroism （CD）, dynamic light scattering （DLS）, fluorescence spectroscopy, and size exclusive chromatography （SEC）. We found that 8 mol·L^-1 urea had no obvious effect on the structure of Mj HSP16.5 at pH 7. The unfolding of Mj HSP16.5 at pH 7 in the presence of guanidine hydrochloride （GdHCI） showed hierarchical behavior. Three significant transitions were observed around 2.0, 3.0, and 6.0 mol·L^-1 GdHC1 at pH 7. ANS （8-anilino-1-naphthalenesulfonic acid） titration results showed that the binding ability of Mj HSP16.5 to ANS decreased gradually as the concentration of GdHC1 increased until around 2.0 mol·L^-1 GdHCl, indicating surface hydrophobic area change, and this first transition was companioned with precipitation of Mj HSP16.5. Acrylamide quenching of fluorescence showed that the Stern-Volmer constant changed at about 3.0 mol ·L^-1 GdHCl, indicating changes of the dimeric interface, and this phase transition was companioned with oligomeric state change from 24-mer to small oligomers （4-mer to 8-mer）. The last unfolding phase started around 5.0 mol·L^-1 GdHCl, with a midpoint of 6.1 mol·L^-1 GdHCl, and Mj HSP16.5 was completely unfolded at 7.0 mol·L^-1 GdHCl. We also found that Mj HSP16.5 could be quite easily unfolded at pH 3, where it could be completely unfolded in 4.0 mol·L^-1 GdHCl.

关 键 词：小热休克蛋白 MJ HSP16.5 蛋白质折叠 变性剂 分级变性 圆二色谱 

分 类 号：Q51[生物学—生物化学]