Mechanisms for GroEL/GroES-mediated folding of a large 86-kDa fusion polypeptide in vitro

Yi Shuian Huang, David T. Chuang

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25 Scopus citations


Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the α subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain α-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-α, when co-expressed with the β subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-α)2β2 structure, similar to the α2β2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-α fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the α moiety on the same chain with rate constants of 1.9 x 10-3 s-1 and 2.95 x 10-4 s-1, respectively. This explained the occurrence of an MBP-α·GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the α-GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-α polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP-α·GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES.

Original languageEnglish (US)
Pages (from-to)10405-10412
Number of pages8
JournalJournal of Biological Chemistry
Issue number15
StatePublished - Apr 9 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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