Encapsulation of an 86-kDa assembly intermediate inside the cavities of GroEL and its single-ring variant SR1 by GRoES

Jiu Li Song, Jun Li, Yi Shuian Huang, David T. Chuang

Research output: Contribution to journalArticle

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Abstract

We described previously that during the assembly of the α2β2 heterotetramer of human mitochondrial branched-chain α-ketoacid dehydrogenase (BCKD), chaperonins GroEL/GroES interact with the kinetically trapped heterodimeric (αβ) 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-8naphthalene-sulfonate. 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-αβ-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-αβ 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.

Original languageEnglish (US)
Pages (from-to)2515-2521
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number4
DOIs
StatePublished - Jan 24 2003

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Encapsulation
Adenosine Triphosphate
Chaperonins
Protein Unfolding
Proteins
Fluorescent Dyes
Gel Chromatography
Conformations
Digestion
Fusion reactions
Gels
Substrates

ASJC Scopus subject areas

  • Biochemistry

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Encapsulation of an 86-kDa assembly intermediate inside the cavities of GroEL and its single-ring variant SR1 by GRoES. / Song, Jiu Li; Li, Jun; Huang, Yi Shuian; Chuang, David T.

In: Journal of Biological Chemistry, Vol. 278, No. 4, 24.01.2003, p. 2515-2521.

Research output: Contribution to journalArticle

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