Structural Studies on Association Mechanism of Chaperonin GroE

来自大肠杆菌的伴侣蛋白Groe是一种热休克蛋白，在介导依赖于ATP的多肽链折叠过程中发挥着重要作用。它由14个L亚基和7个S亚基组成，分别由两个和一个近7重旋转对称的环组成。为了从结构的角度阐明分子伴侣蛋白多亚基组装的作用和形成机制，我对大肠杆菌GroE进行了X射线分析。由于GroE的整个分子似乎很难结晶，所以首次尝试用悬滴气相扩散法结晶一组L亚基。在不同条件下，在3-6个月的时间内获得了两种不同类型的晶体(I型和II型)。在这两种情况下，第一个月的结晶温度设置为4ﾟC，然后改为25ﾟC。通过1.5%蛋白质溶液与15%(w/v)PEG4000(50 mM醋酸盐，pH 5.5)的气相平衡生长出I型晶体。它有一个立方体形状(0.2 mm立方体)。通过0.65%的蛋白质溶液和15%(w/v)的PEG4000(50 mM Tris-HCl，pH 7.5)的气相平衡生长了II型晶体。晶体为板状晶体(0.3 mm×0.2 mm×0.1 mm)。利用KEK-PF的同步辐射进行了X射线衍射实验，KEK-PF晶体具有高达5*分辨率的尖锐反射。

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.