Structural Studies on Association Mechanism of Chaperonin GroE

伴侣蛋白GroE是大肠杆菌的一种热休克蛋白，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，分别形成两个和一个近7重旋转对称环。为了从结构上阐明伴侣蛋白多亚基组装体的功能和形成机制，我对大肠杆菌GroE进行了X射线工作。由于GroE的整个分子似乎很难结晶，因此首次尝试用悬滴气相扩散法结晶L-亚基组装体。在不同的条件下，在3-6个月内获得两种不同类型的晶体（I型和II型）。在这两种情况下，结晶温度在前一个月设定为4 ℃，然后改变为25 ℃。通过1.5%蛋白质溶液相对于15%（w/v）PEG 4000（50 mM乙酸盐，pH 5.5）的蒸气平衡来生长I型晶体。它具有立方体形状（0.2mm立方体）。通过0.65%蛋白质溶液相对于15%（w/v）PEG 4000（50 mM Tris-HCl，pH 7.5）的蒸气平衡来生长II型晶体。它是一个片状晶体（0.3mm x 0.2mm x 0.1mm）。利用同步辐射对KEK-PF晶体进行了X射线衍射实验，后者的衍射峰清晰，分辨率可达5倍。

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.