title: Denaturation and reassembly of chaperonin GroEL studied by solution X-ray scattering.
abstract: 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.
authors.affiliation: Department of Physics, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Munehito
authors.initials: M
authors.lastname: Arai
authors.forename: Tomonao
authors.initials: T
authors.lastname: Inobe
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.forename: Teikichi
authors.initials: T
authors.lastname: Ikura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kihara
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Amemiya
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
mesh_terms: D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation / Q000502:physiology / Q000528:radiation effects; D020673:Protein Renaturation; D014508:Urea; D014965:X-Rays
chemical_list: D018834:Chaperonin 60; D014508:Urea