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1 Department of Physics, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan
3 Department of Physics, Kansai Medical University, Hirakata, Osaka 573-1136, Japan
4 Department of Advanced Materials Science, Graduate School of Frontier Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan
Reprint requests to: Dr. Kunihiro Kuwajima, Department of Physics, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; e-mail: kuwajima{at}phys.s.u-tokyo.ac.jp; fax: 81-3-5841-4512.
We measured the denaturation and reassembly of Escherichia coli chaperonin GroEL using small-angle solution X-ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea-induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil-like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X-ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same structure and the molecular mass as the native GroEL. These results demonstrate that the fully unfolded GroEL monomer can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis and preexisting chaperones. Therefore, GroEL can, in principle, fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under in vivo conditions, in order to avoid misfolding and aggregation.
Keywords: Protein folding; GroEL; molecular chaperone; denaturation; reassembly; X-ray scattering
Abbreviations: ADP, adenosine 5'-diphosphate • ATP, adenosine 5'-triphosphate • CCD, charge-coupled device • GdnHCl, guanidine hydrochloride • I(0), zero-angle scattering intensity • I(Q), scattering intensity at Q • PAGE, polyacrylamide gel electrophoresis • Q, scattering vector • Rg, radius of gyration • SAXS, small-angle X-ray scattering
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