Denatured proteins facilitate the formation of the football-shaped GroEL-(GroES)2 complex

ArticleinBiochemical Journal 427(2):247-54 · April 2010with49 Reads
DOI: 10.1042/BJ20091845 · Source: PubMed
Abstract
Controversy exists over whether the chaperonin GroEL forms a GroEL-(GroES)2 complex (football-shaped complex) during its reaction cycle. We have revealed previously the existence of the football-shaped complex in the chaperonin reaction cycle using a FRET (fluorescence resonance energy transfer) assay [Sameshima, Ueno, Iizuka, Ishii, Terada, Okabe and Funatsu (2008) J. Biol. Chem. 283, 23765-23773]. Although denatured proteins alter the ATPase activity of GroEL and the dynamics of the GroEL-GroES interaction, the effect of denatured proteins on the formation of the football-shaped complex has not been characterized. In the present study, a FRET assay was used to demonstrate that denatured proteins facilitate the formation of the football-shaped complex. The presence of denatured proteins was also found to increase the rate of association of GroES to the trans-ring of GroEL. Furthermore, denatured proteins decrease the inhibitory influence of ADP on ATP-induced association of GroES to the trans-ring of GroEL. From these findings we conclude that denatured proteins facilitate the dissociation of ADP from the trans-ring of GroEL and the concomitant association of ATP and the second GroES.
    • "This would also facilitate the formation of the symmetric complex . We also found that the ATPase activity of GroEL is higher when the levels of the symmetric complex increase [38]. GroEL does not have to employ the maximum ATPase activity in the presence of a small amount of non-native substrate protein. "
    [Show abstract] [Hide abstract] ABSTRACT: The Escherichia coli chaperonin GroEL is an essential molecular chaperone that mediates protein folding in association with its cofactor, GroES. It is widely accepted that GroEL alternates the GroES-sealed folding-active rings during the reaction cycle. In other words, an asymmetric GroEL–GroES complex is formed during the cycle, whereas a symmetric GroEL–(GroES)2 complex is not formed. However, this conventional view has been challenged by the recent reports indicating that such symmetric complexes can be formed in the GroEL–GroES reaction cycle. In this review, we discuss the studies of the symmetric GroEL–(GroES)2 complex, focusing on the molecular mechanism underlying its formation. We also suggest that GroEL can be involved in two types of reaction cycles (asymmetric or symmetric) and the type of cycle used depends on the concentration of non-native substrate proteins.
    Article · Jan 2016
    • "Our data demonstrate that symmetric GroEL:GroES 2 complexes are not significantly populated in the absence of substrate or presence of foldable substrate proteins, in contrast to recent reports171819. However, non-foldable substrate proteins such as LA and -casein can induce the formation of symmetric complexes. "
    [Show abstract] [Hide abstract] ABSTRACT: The chaperonin GroEL, a cylindrical complex consisting of two stacked heptameric rings, and its lid-like cofactor GroES form a nano-cage in which a single polypeptide chain is transiently enclosed and allowed to fold unimpaired by aggregation. GroEL and GroES undergo an ATP-regulated interaction cycle that serves to close and open the folding cage. Recent reports suggest that the presence of non-native substrate protein alters the GroEL/ES reaction by shifting it from asymmetric to symmetric complexes. In the asymmetric reaction mode only one ring of GroEL is GroES-bound and the two rings function sequentially, coupled by negative allostery. In the symmetric mode both GroEL rings are GroES-bound and are folding active simultaneously. Here we find that the results of FRET-based assays recently used to quantify symmetric complexes depend strongly on the fluorophore pair used. We therefore developed a novel assay based on fluorescence cross-correlation spectroscopy to accurately measure GroEL:GroES stoichiometry. This assay avoids fluorophore labeling of GroEL and the use of GroEL cysteine mutants. Our results show that symmetric GroEL:GroES2 complexes are substantially populated only in the presence of non-foldable model proteins, such as α-lactalbumin and α-casein, which 'over-stimulate' the GroEL ATPase and uncouple the negative GroEL inter-ring allostery. In contrast, asymmetric complexes are dominant both in the absence of substrate and presence of foldable substrate proteins. Moreover, uncoupling of the GroEL rings and formation of symmetric GroEL:GroES2 complexes is suppressed at physiological ATP:ADP concentration. We conclude that the asymmetric GroEL:GroES complex represents the main folding active form of the chaperonin. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Apr 2015
    • "In the absence of the denatured protein, either one of the two rings of GroEL binds to GroES [12]. However, in the presence of excess denatured protein, both rings of the chaperonin bind to GroES and assist in the folding of the denatured protein equivalently [13] [14] [15] [16] [17]. Chaperonins greatly improve the yield of protein folding, especially for stringent substrate proteins that tend to form aggregates during spontaneous folding, such as Rubisco and rhodanese, a mitochondrial protein that detoxifies cyanide [4] [18]. "
    [Show abstract] [Hide abstract] ABSTRACT: Protein folding is a biological process that is essential for the proper functioning of proteins in all living organisms. In cells, many proteins require the assistance of molecular chaperones for their folding. Chaperonins belong to a class of molecular chaperones that have been extensively studied. However, the mechanism by which a chaperonin mediates the folding of proteins is still controversial. Denatured proteins are folded in the closed chaperonin cage, leading to the assumption that denatured proteins are completely encapsulated inside the chaperonin cage. In contrast to the assumption, we recently found that denatured protein interacts with hydrophobic residues at the subunit interfaces of the chaperonin, and partially protrude out of the cage. In this review, we will explain our recent results and introduce our model for the mechanism by which chaperonins accelerate protein folding, in view of recent findings.
    Full-text · Article · Apr 2015
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