|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1994 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1993 : ¥1,200,000 (Direct Cost : ¥1,200,000)
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.