Mechanisms for GroEL/GroES-mediated Folding of a Large 86-kDa Fusion Polypeptide in Vitro

Abstract

Our understanding of mechanisms for GroEL/GroESassisted 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 ␣ subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain ␣-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-␣, when co-expressed with the ␤ subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-␣) 2 ␤ 2 structure, similar to the ␣ 2 ␤ 2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/ GroES and Mg 2؉ -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-␣ fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the ␣ moiety on the same chain with rate constants of 1.9 ؋ 10 ؊3 s ؊1 and 2.95 ؋ 10 ؊4 s ؊1 , respectively. This explained the occurrence of an MBP-␣⅐GroEL 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 ␣⅐GroEL 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 -␣ polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP␣⅐GroEL (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.

Interactions of GroEL/GroES with a Heterodimeric Intermediate during α2β2 Assembly of Mitochondrial Branched-chain α-Ketoacid Dehydrogenase

“…It has been suggested that this movement results in doubling the volume of the cis ring cavity of GroEL to accommodate a polypeptide of larger than 70 kDa in size (7). In support of this suggestion, the 75-kDa methylmalonyl-CoA mutase (9), an 82-kDa fusion protein of three tandem green fluorescent proteins (10), and an 86-kDa fusion polypeptide (11) were shown to bind to GroEL, but cis capping of these proteins by GroES did not occur.…”

Mutations that alter the equilibrium between open and closed conformations of Escherichia coli maltose-binding protein impede its ability to enhance the solubility of passenger proteins

“…In fact, Douette et al have presented evidence that MBP and NusA fusion proteins interact with GroEL in E. coli [21]. It is not entirely clear, however, how these rather large fusion proteins could engage the GroEL/GroES chaperone apparatus in the same manner as its natural substrates do, because the size of the chaperone cavity would seem to limit substrates to approximately 50 kDa [22].…”

GroEL/GroES Promote Dissociation/Reassociation Cycles of a Heterodimeric Intermediate during α2β2Protein Assembly

“…Recently, we reported in vitro reconstitution of human BCKD with an absolute requirement for GroEL/GroES and Mg-ATP (30,31); however, the kinetics of BCKD reconstitution was markedly slower than that of chaperonin-mediated refolding of other proteins. A GroEL-␣␤ complex resulting from binding of a large 85-kDa ␣␤ heterodimeric intermediate to GroEL was also isolated.…”