title: Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein.
abstract: Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: So
authors.initials: S
authors.lastname: Ishino
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Tottori University, 4-101 Koyama-Minami, Tottori 680-8552, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kawata
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan, and.
authors.forename: Hideki
authors.initials: H
authors.lastname: Taguchi
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Kajimura
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Matsuzaki
authors.affiliation: From the Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan, hoshi@pharm.kyoto-u.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Hoshino
mesh_terms: D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry* / Q000235:genetics; D002850:Chromatography, Gel; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D049452:Green Fluorescent Proteins
keywords: ATP; Escherichia coli (E. coli); fluorescence; molecular chaperone; protein folding