title: Encapsulation of an 86-kDa assembly intermediate inside the cavities of GroEL and its single-ring variant SR1 by GroES.
abstract: We described previously that during the assembly of the alpha(2)beta(2) heterotetramer of human mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD), chaperonins GroEL/GroES interact with the kinetically trapped heterodimeric (alphabeta) intermediate to facilitate conversion of the latter to the native BCKD heterotetramer. Here, we show that the 86-kDa heterodimeric intermediate possesses a native-like conformation as judged by its binding to a fluorescent probe 1-anilino-8-naphthalenesulfonate. This large heterodimeric intermediate is accommodated as an entity inside cavities of GroEL and its single-ring variant SR1 and is encapsulated by GroES as indicated by the resistance of the heterodimer to tryptic digestion. The SR1-alphabeta-GroES complex is isolated as a stable single species by gel filtration in the presence of Mg-ATP. In contrast, an unfolded BCKD fusion protein of similar size, which also resides in the GroEL or SR1 cavity, is too large to be capped by GroES. The cis-capping mechanism is consistent with the high level of BCKD activity recovered with the GroEL-alphabeta complex, GroES, and Mg-ATP. The 86-kDa native-like heterodimeric intermediate in the BCKD assembly pathway represents the largest protein substrate known to fit inside the GroEL cis cavity underneath GroES, which significantly exceeds the current size limit of 57 kDa established for unfolded proteins.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75390, USA.
authors.forename: Jiu-Li
authors.initials: JL
authors.lastname: Song
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Yi-Shuian
authors.initials: YS
authors.lastname: Huang
authors.forename: David T
authors.initials: DT
authors.lastname: Chuang
mesh_terms: D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D018835:Chaperonin 10 / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D018833:Chaperonins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003720:Densitometry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000494:pharmacology
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D014357:Trypsin; D018833:Chaperonins