Structural Studies on Association Mechanism of Chaperonin GroE

1994 Fiscal Year Final Research Report Summary

• Project/Area Number: 05808062
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Biophysics
• Research Institution: KYOTO UNIVERSITY
• Principal Investigator: HATA Yasuo Kyoto University, Inatitate for Chemical Research, Associate Professor, 化学研究所, 助教授 (10127277)
• Project Period (FY): 1993 – 1994
• Keywords: chaperonin / GroE / GroEL / Crystallization

Research Abstract

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.