Structural Studies on Association Mechanism of Chaperonin GroE

大肠杆菌中的Chaperonin GroE是一种热休克蛋白，在介导atp依赖性多肽链折叠中起重要作用。它由14个l亚基和7个s亚基组成，分别来自两个和一个近7倍旋转对称环。为了从结构的角度阐明伴侣蛋白大型多亚基组装的功能和形成机制，我开展了大肠杆菌GroE的x射线工作。由于很难使整个GroE分子结晶，因此首先尝试用悬垂蒸汽扩散法使l -亚基组合结晶。在不同的条件下，3-6个月得到了两种不同类型的晶体（I型和II型）。在这两种情况下，前一个月的结晶温度都设置为4‰，然后改为25‰。1.5%蛋白质溶液与15% (w/v) PEG4000 （50mM醋酸盐，pH5.5）气相平衡生长出I型晶体。它有一个立方形状（0.2毫米的立方体）。用0.65%的蛋白质溶液与15% (w/v) PEG4000 （50mM Tris-HCl, pH7.5）气相平衡培养II型晶体。它是一个平板晶体（0.3mm x 0.2mm x 0.1mm）。利用KEK-PF同步辐射进行了x射线衍射实验。后一种晶体提供了高达5*分辨率的清晰反射。

Chaperonin GroE from E.coli, a heat-shock protein, Plays an important role in mediating ATP-dependent polypeptide chain folding. It consists of 14 L-subunits and 7 S-subunis which from two and one nearly 7-fold rotationally symmetrical rings, respectively. In order to elucidate the mechanisms of function and formation of large multisubunit assembly of chaperonins from structural viewpoints, I have carried out X-ray work of E.coli GroE.Since it seemed very difficult to crystallize the whole molecule of GroE,a assembly of L-subunits was first tried crystallize by a hanging-drop vapor diffusion method. Two different type of crystals (Type I and Type II) were obtained in 3-6 months under different conditions. In both cases, the crystallization temperature was set to 4ﾟC for the first one monthe and then changed to 25ﾟC.A type I crystal was grown by vapor equilibration of an 1.5% protein solution against 15% (w/v) PEG4000 (50mM acetate, pH5.5) . It had a cubic shape (0.2mm cube) . A Type II crysatal was grown by vapor equilibration of a 0.65% protein solution against 15% (w/v) PEG4000 (50mM Tris-HCl, pH7.5) . It was a plate crystal (0.3mm x 0.2mm x 0.1mm) . X-ray diffraction experiments were performed by using synchrotron radiation of KEK-PF.The latter crystal gave sharp reflections up to 5* resolution.