On the role of symmetrical and asymmetrical chaperonin complexes in assisted protein folding.

The cylindrical chaperonin GroEL of E. coli and its ring-shaped cofactor GroES cooperate in mediating the ATP-dependent folding of a wide range of polypeptides in vivo and in vitro. By binding to the ends of the GroEL cylinder, GroES displaces GroEL-bound polypeptide into an enclosed folding cage, thereby preventing protein aggregation during folding. The dynamic interaction of GroEL and GroES is regulated by the GroEL ATPase and involves the formation of asymmetrical GroEL:GroES1 and symmetrical GroEL: GroES2 complexes. The proposed role of the symmetrical complex as a catalytic intermediate of the chaperonin mechanism has been controversial. It has also been suggested that the formation of GroEL:GroES2 complexes allows the folding of two polypeptide molecules per GroEL reaction cycle, one in each ring of GroEL. By making use of a procedure to stabilize chaperonin complexes by rapid crosslinking for subsequent analysis by native PAGE, we have quantified the occurrence of GroEL:GroES1 and GroEL:GroES2 complexes in active refolding reactions under a variety of conditions using mitochondrial malate dehydrogenase (mMDH) as a substrate. Our results show that the symmetrical complexes are neither required for chaperonin function nor does their presence significantly increase the rate of mMDH refolding. In contrast, chaperonin-assisted folding is strictly dependent on the formation of asymmetrical GroEL:GroES1 complexes. These findings support the view that GroEL:GroES2 complexes have no essential role in the chaperonin mechanism.     

GroEL channels the folding of thioredoxin along one kinetic route.

Many proteins display complex folding kinetics, which represent multiple parallel folding pathways emanating from multiple unfolded forms and converging to the unique native form. The small protein thioredoxin from Escherichia coli is one such protein. The effect of the chaperonin GroEL on modulating the complex energy landscape that separates the unfolded ensemble from the native state of thioredoxin has been studied. It is shown that while the fluorescence change accompanying folding occurs in five kinetic phases in the absence of GroEL, only the two slowest kinetic phases are discernible in the presence of saturating concentrations of GroEL. This result is shown to be consistent with only one out of several available folding routes being operational in the presence of GroEL. It is shown that native protein, which forms via fast as well as slow routes in the absence of GroEL, forms only via a slow route in its presence. The effect of GroEL on the folding of thioredoxin is shown to be the consequence of it binding differentially to the many folding-competent forms. While some of these forms can continue folding when bound to GroEL, others cannot. All molecules are then drawn into the operational folding route by the law of mass action. This observation indicates a new role for GroEL, which is to bias the energy landscape of a folding polypeptide towards fewer available pathways. It is suggested that such channeling might be a mechanism to avoid possible aggregation-prone routes available to a refolding polypeptide in vivo.     

Chaperones GroEL/GroES Accelerate the Refolding of a Multidomain Protein through Modulating On-pathway Intermediates

Despite a vast amount information on the interplay of GroEL, GroES, and ATP in chaperone-assisted folding, the molecular details on the conformational dynamics of folding polypeptide during its GroEL/GroES-assisted folding cycle is quite limited. Practically no such studies have been reported to date on large proteins, which often have difficulty folding in vitro. The effect of the GroEL/GroES chaperonin system on the folding pathway of an 82-kDa slow folding protein, malate synthase G (MSG), was investigated. GroEL bound to the burst phase intermediate of MSG and accelerated the slowest kinetic phase associated with the formation of native topology in the spontaneous folding pathway. GroEL slowly induced conformational changes on the bound burst phase intermediate, which was then transformed into a more folding-compatible form. Subsequent addition of ATP or GroES/ATP to the GroEL-MSG complex led to the formation of the native state via a compact intermediate with the rate several times faster than that of spontaneous refolding. The presence of GroES doubled the ATP-dependent reactivation rate of bound MSG by preventing multiple cycles of its GroEL binding and release. Because GroES bound to the trans side of GroEL-MSG complex, it may be anticipated that confinement of the substrate underneath the co-chaperone is not required for accelerating the rate in the assisted folding pathway. The potential role of GroEL/GroES in assisted folding is most likely to modulate the conformation of MSG intermediates that can fold faster and thereby eliminate the possibility of partial aggregation caused by the slow folding intermediates during its spontaneous refolding pathway.     

Active Cage Mechanism of Chaperonin-Assisted Protein Folding Demonstrated at Single-Molecule Level

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations (100 pM) to exclude aggregation, we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP) by single-pair fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We find that GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. Accelerated folding is achieved by encapsulation of folding intermediate in the GroEL/ES cage, independent of repetitive cycles of protein binding and release from GroEL. Moreover, photoinduced electron transfer experiments provided direct physical evidence that the confining environment of the chaperonin restricts polypeptide chain dynamics. This effect is mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. EL(KKK2)/ES functions as a passive cage in which folding occurs at the slow spontaneous rate. Taken together our findings suggest that protein encapsulation can accelerate folding by entropically destabilizing folding intermediates, in strong support of an active chaperonin mechanism in the folding of some proteins. Accelerated folding is biologically significant as it adjusts folding rates relative to the speed of protein synthesis.     

Double mutant MBP refolds at same rate in free solution as inside the GroEL/GroES chaperonin chamber when aggregation in free solution is prevented

Under "permissive" conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5-10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering. When refolded in chloride-free buffer, however, dynamic light scattering was eliminated, refolding became concentration-independent, and the rate of refolding became the same as that in GroEL/GroES. The GroEL/GroES chamber thus appears to function passively toward DM-MBP.     

Refolding of target proteins from a "rigid" mutant chaperonin demonstrates a minimal mechanism of chaperonin binding and release

One of the most interesting facets of GroEL-facilitated protein folding lies in the fact that the requirement for a successful folding reaction of a given protein target depends upon the refolding conditions used. In this report, we utilize a mutant of GroEL (GroEL T89W) whose domain movements have been drastically restricted, producing a chaperonin that is incapable of utilizing the conventional cyclic mechanism of chaperonin action. This mutant was, however, still capable of improving the refolding yield of lactate dehydrogenase in the absence of both GroES and ATP hydrolysis. A very rapid interconversion of conformations was detected in the mutant immediately after ATP binding, and this interconversion was inferred to form part of the target release mechanism in this mutant. The possibility exists that some target proteins, although dependent on GroEL for improved refolding yields, are capable of refolding successfully by utilizing only portions of the entire mechanism provided by the chaperonins.     

Binding of a chaperonin to the folding intermediates of lactate dehydrogenase.

When Bacillus stearothermophilus LDH dimer is incubated with increasing concentrations of the denaturant guanidinium chloride, three distinct unfolded states of the molecule are observed at equilibrium [Smith, C. J., et al. (1991) Biochemistry 30, 1028-1036]. The kinetics of LDH refolding are consistent with an unbranched progression through these states. The Escherichia coli chaperonin, GroEL, binds with high affinity to the completely denatured form and more weakly to the earliest folding intermediate, thus retarding the refolding process. A later structurally defined folding intermediate, corresponding to a molten globule form, is not bound by GroEL; neither is the inactive monomer. The complex between GroEL and denatured LDH is destabilized by the binding of magnesium/ATP (Mg/ATP) or by the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP). From our initial kinetic data, we propose that GroEL exists in two interconvertible forms, one of which is stabilized by the binding of Mg/ATP but associates weakly with the unfolded protein. The other is destabilized by Mg/ATP and associates strongly with unfolded LDH. The relevance of these findings to the role of GroEL in vivo is discussed.     

Capture of monomeric refolding intermediate of human muscle creatine kinase

Human muscle creatine kinase (CK) is an enzyme that plays an important physiological role in the energy metabolism of humans. It also serves as a typical model for studying refolding of proteins. A study of the refolding and reactivation process of guanidine chloride-denatured human muscle CK is described in the present article. The results show that the refolding process can be divided into fast and slow folding phases and that an aggregation process competes with the proper refolding process at high enzyme concentration and high temperature. An intermediate in the early stage of refolding was captured by specific protein molecules: the molecular chaperonin GroEL and alpha(s)-casein. This intermediate was found to be a monomer, which resembles the "molten globule" state in the CK folding pathway. To our knowledge, this is the first monomeric intermediate captured during refolding of CK. We propose that aggregation is caused by interaction between such monomeric intermediates. Binding of GroEL with this intermediate prevents formation of aggregates by decreasing the concentration of free monomeric intermediates, whereas binding of alpha(s)-casein with this intermediate induces more aggregation.     

High salt-induced conversion of Escherichia coli GroEL into a fully functional thermophilic chaperonin

The GroE chaperonin system can adapt to and function at various environmental folding conditions. To examine chaperonin-assisted protein folding at high salt concentrations, we characterized Escherichia coli GroE chaperonin activity in 1.2 m ammonium sulfate. Our data are consistent with GroEL undergoing a conformational change at this salt concentration, characterized by elevated ATPase activity and increased exposure of hydrophobic surface, as indicated by increased binding of the fluorophore bis-(5, 5')-8-anilino-1-naphthalene sulfonic acid to the chaperonin. The presence of the salt results in increased substrate stringency and dependence on the full GroE system for release and productive folding of substrate proteins. Surprisingly, GroEL is fully functional as a thermophilic chaperonin in high concentrations of ammonium sulfate and is stable at temperatures up to 75 degrees C. At these extreme conditions, GroEL can suppress aggregation and mediate refolding of non-native proteins.     

GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism.

The chaperonin GroEL binds folding intermediates of four-disulfidehen lysozyme transiently within its central cavity. Using stopped flow fluorescence we show that GroEL binds early intermediates in folding and accelerates the slow kinetic phase that reflects the reversal of non-native interactions involving tryptophan residues and the formation of the native state. Pulsed hydrogen exchange monitored by electrospray ionization mass spectrometry demonstrates that GroEL does not alter the folding mechanism, nor are protected species unfolded by the chaperonin. The data suggest a mechanism for GroEL-assisted folding in which the reorganization of non-native tertiary interactions is facilitated but domain folding is unperturbed.     

The interaction of the GroEL chaperone with early kinetic intermediates of renaturing proteins inhibits the formation of their native structure

The main function of the chaperone GroEL is to prevent nonspecific association of nonnative protein chains and provide their correct folding. In the present work, the renaturation kinetics of three globular proteins (human alpha-lactalbumin, bovine carbonic anhydrase, and yeast phosphoglycerate kinase) in the presence of different molar excess of GroEL (up to 10-fold) was studied. It was shown that the formation of the native structure during the refolding of these proteins is retarded with an increase in GroEL molar excess due to the interaction of kinetic protein intermediates with the chaperone. Mg(2+)-ATP and Mg(2+)-ADP weaken this interaction and decrease the retarding effect of GroEL on the protein refolding kinetics. The theoretical modeling of protein folding in the presence of GroEL showed that the experimentally observed linear increase in the protein refolding half-time with increasing molar excess of GroEL must occur only when the protein adopts its native structure outside of GroEL (i.e. in the free state), while the refolding of the protein in the complex with GroEL is inhibited. The dissociation constants of GroEL complexed with the kinetic intermediates of the proteins studied were evaluated, and a simple mechanism of the functioning of GroEL as a molecular chaperone was proposed.     

Influence of the GroE molecular chaperone machine on the in vitro refolding of Escherichia coli beta-galactosidase.

We have studied the effect of the components of the GroE molecular chaperone machine on the refolding of the Escherichia coli enzyme beta-galactosidase, a tetrameric protein whose 116-kDa promoters should not completely fit within the central cavity of the GroEL toroid. In the absence of other additives, GroEL formed a weak complex with chemically denatured beta-galactosidase, reduced its propensity to aggregate, and increased the recovery yields of active enzyme twofold without altering its folding pathway. When present together with the chaperonin, ATP--and to a lesser extent AMP-PNP--reduced the recovery yields and led to the resumption of aggregation. The use of the complete chaperonin system (GroEL, GroES, and ATP) eliminated the GroEL-mediated increase in recovery and folding proceeded less efficiently than in buffer alone. This unusual behavior can be explained in terms of a chaperonin "buffering" effect and the different affinities of GroE complexes for denatured beta-galactosidase.     

The intermediate domain defines broad nucleotide selectivity for protein folding in Chlamydophila GroEL1

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln(400) and Ile(404) in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.     

Do Chaperonins Boost Protein Yields by Accelerating Folding or Preventing Aggregation?

The GroEL chaperonin has the ability to behave as an unfoldase, repeatedly denaturing proteins upon binding, which in turn can free them from kinetic traps and increase their folding rates. The complex formed by GroEL+GroES+ATP can also act as an infinite dilution cage, enclosing proteins within a protective container where they can fold without danger of aggregation. Controversy remains over which of these two properties is more critical to the GroEL/ES chaperonin's function. We probe the importance of the unfoldase nature of GroEL under conditions where aggregation is the predominant protein degradation pathway. We consider the effect of a hypothetical mutation to GroEL which increases the cycle frequency of GroEL/ES by increasing the rate of hydrolysis of GroEL-bound ATP. Using a simple kinetic model, we show that this modified chaperonin would be self-defeating: any potential reduction in folding time would be negated by an increase in time spent in the bulk, causing an increase in aggregation and a net decrease in protein folding yields.     

Requirement for GroEL/GroES-dependent protein folding under nonpermissive conditions of macromolecular crowding

Macromolecular crowding is a critical parameter affecting the efficiency of cellular protein folding. Here we show that the proteins dihydrofolate reductase, enolase, and green fluorescent protein, which can fold spontaneously in diluted buffer, lose this ability in a crowded environment. Instead, they accumulate as soluble, protease-sensitive non-native species. Their folding becomes dependent on the complete GroEL/GroES chaperonin system and is not affected by trap-GroEL, indicating that folding has to occur in the chaperonin cavity with release of nativelike proteins into the bulk solution. In addition, we demonstrate that efficient folding in the chaperonin cavity requires ATP hydrolysis, as formation of ternary GroEL/GroES complexes with substrate proteins in the presence of ADP results only in very inefficient reactivation. However, protein refolding reactions using ADP-fluoroaluminate complexes, or single-ring GroEL and GroES under conditions where only a single round of ATP hydrolysis occurs, yield large amounts of refolded enzymes. Thus, the mode of initial ternary complex formation appears to be critical for subsequent productive release of substrate into the cavity under certain crowding conditions, and is only efficient when triggered by ATP hydrolysis. Our data indicate that stringent conditions of crowding can impart a stronger dependence of folding proteins on the assistance by chaperonins.     

Essential role of the chaperonin folding compartment in vivo

The GroEL/GroES chaperonin system of Escherichia coli forms a nano-cage allowing single protein molecules to fold in isolation. However, as the chaperonin can also mediate folding independently of substrate encapsulation, it remained unclear whether the folding cage is essential in vivo. To address this question, we replaced wild-type GroEL with mutants of GroEL having either a reduced cage volume or altered charge properties of the cage wall. A stepwise reduction in cage size resulted in a gradual loss of cell viability, although the mutants bound non-native protein efficiently. Strikingly, a mild reduction in cage size increased the yield and the apparent rate of green fluorescent protein folding, consistent with the view that an effect of steric confinement can accelerate folding. As shown in vitro, the observed acceleration of folding was dependent on protein encapsulation by GroES but independent of GroES cycling regulated by the GroEL ATPase. Altering the net-negative charge of the GroEL cage wall also strongly affected chaperonin function. Based on these findings, the GroEL/GroES compartment is essential for protein folding in vivo.     

New aspects on the mechanism of GroEL-assisted protein folding

The mechanism of assisted protein folding by the chaperonin GroEL alone or in complex with the co-chaperonin GroES and in the presence or absence of nucleotides has been subject to extensive investigations during the last years. In this paper we present data where we have inactivated GroEL by stepwise blocking the nucleotide binding sites using the non-hydrolyzable ATP analogue, (Cr(H2O)4)3+ATP. We correlated the amount of accessible nucleotide binding sites with the residual ATP hydrolysis activity of GroEL as well as the residual refolding activity for two different model substrates. Under the conditions used, folding of the substrate proteins and ATP hydrolysis were directly proportional to the residual, accessible nucleotide binding sites. In the presence of GroES, 50% of the nucleotide binding sites were protected from inactivation by CrATP and the resulting protein retains 50% of both ATPase and refolding activity. The results strongly suggest that under the conditions used in our experiments, the nucleotide binding sites are additive in character and that by blocking of a certain number of binding sites a proportional amount of ATP hydrolysis and refolding activities are inactivated. The experiments including GroES suggest that full catalytic activity of GroEL requires both rings of the chaperonin. Blocking of the nucleotide binding sites of one ring still allows function of the second ring.     

Designing a High Throughput Refolding Array Using a Combination of the GroEL Chaperonin and Osmolytes

Although GroE chaperonins and osmolytes had been used separately as protein folding aids, combining these two methods provides a considerable advantage for folding proteins that cannot fold with either osmolytes or chaperonins alone. This technique rapidly identifies superior folding solution conditions for a broad array of proteins that are difficult or impossible to fold by other methods. While testing the broad applicability of this technique, we have discovered that osmolytes greatly simplify the chaperonin reaction by eliminating the requirement for the co-chaperonin GroES which is normally involved in encapsulating folding proteins within the GroEL–GroES cavity. Therefore, combinations of soluble or immobilized GroEL, osmolytes and ATP or even ADP are sufficient to refold the test proteins. The first step in the chaperonin/osmolyte process is to form a stable long-lived chaperonin–substrate protein complex in the absence of nucleotide. In the second step, different osmolyte solutions are added along with nucleotides, thus forming a ‘folding array’ to identify superior folding conditions. The stable chaperonin–substrate protein complex can be concentrated or immobilized prior to osmolyte addition. This procedure prevents-off pathway aggregation during folding/refolding reactions and more importantly allows one to refold proteins at concentrations (~mg/ml) that are substantially higher than the critical aggregation concentration for given protein. This technique can be used for successful refolding of proteins from purified inclusion bodies. Recently, other investigators have used our chaperonin/osmolyte method to demonstrate that a mutant protein that misfolds in human disease can be rescued by GroEL/osmolyte system. Soluble or immobilized GroEL can be easily removed from the released folded protein using simple separation techniques. The method allows for isolation of folded monomeric or oligomeric proteins in quantities sufficient for X-ray crystallography or NMR structural determinations.     

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 Can Unfold Late Intermediates Populated on the Folding Pathways of Monellin

The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.