Binding, encapsulation and ejection: substrate dynamics during a chaperonin-assisted folding reaction

Mitochondrial malate dehydrogenase (mMDH) folds more rapidly in the presence of GroEL, GroES and ATP than it does unassisted. The increase in folding rate as a function of the concentration of GroEL-ES reaches a maximum at a stoichiometry which is approximately equimolar (mMDH subunits:GroEL oligomer) and with an apparent dissociation constant K' for the GroE acceptor state of at least 1 x 10(-8) M. However, even at chaperonin concentrations which are 4000 x K', i.e. at negligible concentrations of free mMDH, the observed folding rate of the substrate remains at its optimum, showing not only that folding occurs in the chaperonin-mMDH complex but also that this rate is uninhibited by any interactions with sites on GroEL. Despite the ability of mMDH to fold on the chaperonin, trapping experiments show that its dwell time on the complex is only 20 seconds. This correlates with both the rate of ATP turnover and the dwell time of GroES on the complex and is only approximately 5% of the time taken for the substrate to commit to the folded state. The results imply that ATP drives the chaperonin complex through a cycle of three functional states: (1) an acceptor complex in which the unfolded substrate is bound tightly; (2) an encapsulation state in which it is sequestered but direct protein-protein contact is lost so that folding can proceed unhindered; and (3) an ejector state which forces dissociation of the substrate whether folded or not.     

Characterization of a functional GroEL14(GroES7)2 chaperonin hetero-oligomer

Chaperonins GroEL and GroES form two types of hetero-oligomers in vitro that can mediate the folding of proteins. Chemical cross-linking and electron microscopy showed that in the presence of adenosine triphosphate (ATP), two GroES7 rings can successively bind a single GroEL14 core oligomer. The symmetric GroEL14(GroES7)2 chaperonin, whose central cavity appears obstructed by two GroES7 rings, can nonetheless stably bind and assist the ATP-dependent refolding of RuBisCO enzyme. Thus, unfolded proteins first bind and possibly fold on the external envelope of the chaperonin hetero-oligomer.     

Asymmetrical interaction of GroEL and GroES in the ATPase cycle of assisted protein folding

The chaperonins GroEL and GroES of Escherichia coli facilitate protein folding in an adenosine triphosphate (ATP)-dependent reaction cycle. The kinetic parameters for the formation and dissociation of GroEL-GroES complexes were analyzed by surface plasmon resonance. Association of GroES and subsequent ATP hydrolysis in the interacting GroEL toroid resulted in the formation of a stable GroEL:ADP:GroES complex. The complex dissociated as a result of ATP hydrolysis in the opposite GroEL toroid, without formation of a symmetrical GroEL:(GroES)2 intermediate. Dissociation was accelerated by the addition of unfolded polypeptide. Thus, the functional chaperonin unit is an asymmetrical GroEL:GroES complex, and substrate protein plays an active role in modulating the chaperonin reaction cycle.     

Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.     

GroEL-GroES-mediated protein folding requires an intact central cavity

The chaperonin GroEL is an oligomeric double ring structure that, together with the cochaperonin GroES, assists protein folding. Biochemical analyses indicate that folding occurs in a cis ternary complex in which substrate is sequestered within the GroEL central cavity underneath GroES. Recently, however, studies of GroEL "minichaperones" containing only the apical substrate binding subdomain have questioned the functional importance of substrate encapsulation within GroEL-GroES complexes. Minichaperones were reported to assist folding despite the fact that they are monomeric and therefore cannot form a central cavity. Here we compare directly the folding activity of minichaperones with that of the full GroEL-GroES system. In agreement with earlier studies, minichaperones assist folding of some proteins. However, this effect is observed only under conditions where substantial spontaneous folding is also observed and is indistinguishable from that resulting from addition of the nonchaperone protein alpha-casein. By contrast, the full GroE system efficiently promotes folding of several substrates under conditions where essentially no spontaneous folding is observed. These data argue that the full GroEL folding activity requires the intact GroEL-GroES complex, and in light of previous studies, underscore the importance of substrate encapsulation for providing a folding environment distinct from the bulk solution.     

Role of the GroEL chaperonin intermediate domain in coupling ATP hydrolysis to polypeptide release

Modification of the Escherichia coli chaperonin GroEL with N-ethylmaleimide at residue Cys138 affects the structural and functional integrity of the complex. Nucleotide affinity and ATPase activity of the modified chaperonin are increased, whereas cooperativity of ATP hydrolysis and affinity for GroES are reduced. As a consequence, release and folding of substrate proteins are strongly impaired and uncoupled from ATP hydrolysis in a temperature-dependent manner. Folding of dihydrofolate reductase at 25 degrees C becomes dependent on GroES, whereas folding of typically GroES-dependent proteins is blocked completely. At 37 degrees C, GroES binding is restored to normal levels, and the modified GroEL regains its chaperone activity to some extent. These results assign a central role to the intermediate GroEL domain for transmitting conformational changes between apical and central domains, and for coupling ATP hydrolysis to productive protein release.     

One founder/one gene hypothesis in a new expanding population: Saguenay (Quebec, Canada)

Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.     

Protein folding: how the mechanism of GroEL action is defined by kinetics

We propose a mechanism for the role of the bacterial chaperonin GroEL in folding proteins. The principal assumptions of the mechanism are (i) that many unfolded proteins bind to GroEL because GroEL preferentially binds small unstructured regions of the substrate protein, (ii) that substrate protein within the cavity of GroEL folds by the same kinetic mechanism and rate processes as in bulk solution, (iii) that stable or transient complexes with GroEL during the folding process are defined by a kinetic partitioning between formation and dissociation of the complex and the rate of folding and unfolding of the protein, and (iv) that dissociation from the complex in early stages of folding may lead to aggregation but dissociation at a late stage leads to correct folding. The experimental conditions for refolding may play a role in defining the function of GroEL in the folding pathway. We propose that the role of GroES and MgATP, either binding or hydrolysis, is to regulate the association and dissociation processes rather than affecting the rate of folding.     

SCL 90-R interpretation and brain tumour: a correction factor?

Two models are being considered for the mechanism of chaperonin-assisted protein folding in E. coli: (i) GroEL/GroES act primarily by enclosing substrate polypeptide in a folding cage in which aggregation is prevented during folding. (ii) GroEL mediates the repetitive unfolding of misfolded polypeptides, returning them onto a productive folding track. Both models are not mutually exclusive, but studies with the polypeptide-binding domain of GroEL have suggested that unfolding is the primary mechanism, enclosure being unnecessary. Here we investigate the capacity of the isolated apical polypeptide-binding domain to functionally replace the complete GroEL/GroES system. We show that the apical domain binds aggregation-sensitive polypeptides but cannot significantly assist their refolding in vitro and fails to replace the groEL gene or to complement defects of groEL mutants in vivo. A single-ring version of GroEL cannot substitute for GroEL. These results strongly support the view that sequestration of aggregation-prone intermediates in a folding cage is an important element of the chaperonin mechanism.     

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.     

Differential expression of calbindin and calmodulin in motoneurons after hypoglossal axotomy

Interferon-gamma (IFN-gamma) is a structurally labile cytokine that rapidly denatures upon exposure to acid or heat. Here we show that both acid-denatured (pH 2) and thermally inactivated (50 degrees C) porcine IFN-gamma can be rescued with the Escherichia coli GroEL/ES chaperonin system and ATP, and reassembled into bioactive dimers. At 35 degrees C, spontaneous refolding of acid-denatured IFN-gamma was found to be dependent on the presence of guanidinium hydrochloride (0.15-0.25 M) or NaCl (0.1-0.2 M). Under non-permissive reaction conditions for regain of native structure (low-ionic-strength buffer at 35 degrees C), the yield of IFN-gamma refolded with GroEL/ES/ATP increased about 30-fold above the level of spontaneous refolding. In the absence of GroES, GroEL captured IFN-gamma in a folding-competent complex. Under these conditions, both ATP and alpha-casein induced release of IFN-gamma from GroEL but with the released protein tending to partition into sedimentable aggregates. Only in the presence of GroES, did ATP induce complete discharge of IFN-gamma from GroEL, with the released protein refolded into a conformation that is (a) immunoreactive/bio-active, (b) resistant to precipitation and (c) in a dimeric configuration. Chicken egg albumin and 90-kDa heat-shock protein were inactive in the exertion of any protective effect against physicochemical stress. The precise amount of protein refolded to the native state at different times of the folding reaction was determined by alpha-casein quenching and ELISA. The former is based on the conversion by excess alpha-casein of any population of unfolded IFN-gamma into one that escapes antibody recognition by subsequent ELISA. Since the native dimers, however, are not affected by alpha-casein quenching, immunoreactivity is directly proportional to the yield of correctly refolded protein. The validity of this approach was confirmed by measurement of biological activity. GroEL/ES-meditated reactivation amounted to > 80% both by ELISA and antiviral assay.     

The ins and outs of a molecular chaperone machine

Genetic and biochemical work has highlighted the biological importance of the GroEL/GroES (Hsp60/Hsp10; cpn60/cpn10) chaperone machine in protein folding. GroEL's donut-shaped structure has attracted the attention of structural biologists because of its elegance as well as the secrets (substrates) it can hide. The recent determination of the GroES and GroEL/GroES structures provides a glimpse of their plasticity, revealing dramatic conformational changes that point to an elaborate mechanism, coupling ATP hydrolysis to substrate release by GroEL.     

GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms

The chaperonin GroEL is a ribosome-sized double-ring structure that assists in folding a diverse set of polypeptides. We have examined the fate of a polypeptide during a chaperonin-mediated folding reaction. Strikingly, we find that, upon addition of ATP and the cochaperonin GroES, polypeptide is released rapidly from GroEL in a predominantly nonnative conformation that can be trapped by mutant forms of GroEL that are capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while the remainder is rebound rapidly by other GroEL molecules. Folding appears to occur in an all-or-none manner, as proteolysis and tryptophan fluorescence indicate that after rebinding, polypeptide has the same structure as in the original complex. These observations suggest that GroEL functions by carrying out multiple rounds of binding aggregation-prone or kinetically trapped intermediates, maintaining them in an unfolded state, and releasing them to attempt to fold in solution.     

Chaperonin GroE-facilitated refolding of disulfide-bonded and reduced Taka-amylase A from Aspergillus oryzae

The refolding characteristics of Taka-amylase A (TAA) from Aspergillus oryzae in the presence of the chaperonin GroE were studied in terms of activity and fluorescence. Disulfide-bonded (intact) TAA and non-disulfide-bonded (reduced) TAA were unfolded in guanidine hydrochloride and refolded by dilution into buffer containing GroE. The intermediates of both intact and reduced enzymes were trapped by GroEL in the absence of nucleotide. Upon addition of nucleotides such as ATP, ADP, CTP or UTP, the intermediates were released from GroEL and recovery of activity was detected. In both cases, the refolding yields in the presence of GroEL and ATP were higher than spontaneous recoveries. Fluorescence studies of intrinsic tryptophan and a hydrophobic probe, 8-anilinonaphthalene-1-sulfonate, suggested that the intermediates trapped by GroEL assumed conformations with different hydrophobic properties. The presence of protein disulfide isomerase or reduced and oxidized forms of glutathione in addition to GroE greatly enhanced the refolding reaction of reduced TAA. These findings suggest that GroE has an ability to recognize folding intermediates of TAA protein and facilitate refolding, regardless of the existence or absence of disulfide bonds in the protein.     

Partitioning of rhodanese onto GroEL. Chaperonin binds a reversibly oxidized form derived from the native protein

The mammalian mitochondrial enzyme, rhodanese, can form stable complexes with the Escherichia coli chaperonin GroEL if it is either refolded from 8 M urea in the presence of chaperonin or is simply added to the chaperonin as the folded conformer at 37 degreesC. In the presence of GroEL, the kinetic profile of the inactivation of native rhodanese followed a single exponential decay. Initially, the inactivation rates showed a dependence on the chaperonin concentration but reached a constant maximum value as the GroEL concentration increased. Over the same time period, in the absence of GroEL, native rhodanese showed only a small decline in activity. The addition of a non-denaturing concentration of urea accelerated the inactivation and partitioning of rhodanese onto GroEL. These results suggest that the GroEL chaperonin may facilitate protein unfolding indirectly by interacting with intermediates that exist in equilibrium with native rhodanese. The activity of GroEL-bound rhodanese can be completely recovered upon addition of GroES and ATP. The reactivation kinetics and commitment rates for GroEL-rhodanese complexes prepared from either unfolded or native rhodanese were identical. However, when rhodanese was allowed to inactivate spontaneously in the absence of GroEL, no recovery of activity was observed upon addition of GroEL, GroES, and ATP. Interestingly, the partitioning of rhodanese and its subsequent inactivation did not occur when native rhodanese and GroEL were incubated under anaerobic conditions. Thus, our results strongly suggest that the inactive intermediate that partitions onto GroEL is the reversibly oxidized form of rhodanese.     

The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network

The role of small heat-shock proteins in Escherichia coli is still enigmatic. We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones. IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress. While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner. Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates. Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme. IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins.     

Preventing mouth injuries during sports

In the presence of MgATP or MgADP the E. coli chaperonin proteins, GroEL and GroES, form a stable asymmetric complex with a stoichiometry of two GroEL7:one GroES7: seven MgADP. The distribution of the ligands between the two heptameric GroEL rings is crucial to our understanding of the mechanism of chaperonin-assisted folding, being either cis (i.e. [GroEL7.MgADP7.GroES7]-[GroEL7]) or trans (i.e. [GroEL7.MgADP7]-[GroEL7.GroES7]. On the basis of cross-linking experiments with 8-azido-ATP and the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), it was suggested that GroES and MgADP are bound to the same GroEL ring which resists proteinase K digestion [Nature 366 (1993) 228-233]. However, we find that the SPDP-promoted cross linking of GroES and GroEL occurs in the absence of Mg2+, ADP or ATP, which are required for the formation of the asymmetric complex. Cross-linking is shown to occur only when the SPDP-modified GroES is co-precipitated with GroEL by trichloracetic acid. Furthermore, there are structural grounds for questioning whether SPDP can crosslink, in a physiologically relevant manner, an amino group of GroES with any of the cysteinyl groups of GroEL.     

Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL

The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel beta-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.     

The chaperonin cycle cannot substitute for prolyl isomerase activity, but GroEL alone promotes productive folding of a cyclophilin-sensitive substrate to a cyclophilin-resistant form

The chaperonin GroEL and the peptidyl-prolyl cis-trans isomerase cyclophilin are major representatives of two distinct cellular systems that help proteins to adopt their native three-dimensional structure: molecular chaperones and folding catalysts. Little is known about whether and how these proteins cooperate in protein folding. In this study, we have examined the action of GroEL and cyclophilin on a substrate protein in two distinct prolyl isomerization states. Our results indicate that: (i) GroEL binds the same substrate in different prolyl isomerization states. (ii) GroEL-ES does not promote prolyl isomerizations, but even retards isomerizations. (iii) Cyclophilin cannot promote the correct isomerization of prolyl bonds of a GroEL-bound substrate, but acts sequentially after release of the substrate from GroEL. (iv) A denatured substrate with all-native prolyl bonds is delayed in folding by cyclophilin due to isomerization to non-native prolyl bonds; a substrate that has proceeded in folding beyond a stage where it can be bound by GroEL is still sensitive to cyclophilin. (v) If a denatured cyclophilin-sensitive substrate is first bound to GroEL, however, productive folding to a cyclophilin-resistant form can be promoted, even without GroES. We conclude that GroEL and cyclophilin act sequentially and exert complementary functions in protein folding.     

Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements

The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially-folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (phi = 18.6 ns) was determined for partially-folded species tagged with anthraniloyl. GroEL recognizes partially-folded species with a K(D) approximately 60 nM. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, approaches a value of 280 ns in the absence of ATP. Reactivation of mMDH-ANT by addition of GroEL and ATP brings about a significant decrease in the observed rotational correlation time. The results indicate that partially-folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.