GroEL-assisted dehydrogenase folding mediated by coenzyme is ATP-independent.

It has been commonly accepted that GroEL functions as a chaperone by modulation of its affinity for folding intermediates through binding and hydrolysis of ATP. However, we have found that NAD, as a coenzyme of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), also stimulates the discharge of GAPDH folding intermediate from its stable complex with GroEL formed in the absence of ATP and assists refolding with the same yield as ATP/Mg(2+) does. The reactivation further increases when ATP is also present, but addition of Mg(2+) has no more effect. NADP, a coenzyme of glucose-6-phosphate dehydrogenase, also releases its folding intermediates from GroEL and increases reactivation. Different from ATP, NAD triggers the release of GAPDH intermediates bound by GroEL via binding with GAPDH itself but not with GroEL, and the released intermediates all folded to native molecules without the formation of aggregation. The collaborative effects of coenzyme and GroEL mediate GroEL-assisted dehydrogenase folding in an ATP-independent way.     

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.     

Thermodynamics of the folding of D-glyceraldehyde-3-phosphate dehydrogenase assisted by protein disulfide isomerase studied by microcalorimetry.

Thermodynamics of the refolding of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assisted by protein disulfide isomerase (PDI), a molecular chaperone, has been studied by isothermal microcalorimetry at different molar ratios of PDI/GAPDH and temperatures using two thermodynamic models proposed for chaperone-substrate binding and chaperone-assisted substrate folding, respectively. The binding of GAPDH folding intermediates to PDI is driven by a large favorable enthalpy decrease with a large unfavorable entropy reduction, and shows strong enthalpy-entropy compensation and weak temperature dependence of Gibbs free energy change. A large negative heat-capacity change of the binding, -156 kJ.mol(-1).K(-1), at all temperatures examined indicates that hydrophobic interaction is a major force for the binding. The binding stoichiometry shows one dimeric GAPDH intermediate per PDI monomer. The refolding of GAPDH assisted by PDI is a largely exothermic reaction at 15.0-25.0 degrees C. With increasing temperature from 15.0 to 37.0 degrees C, the PDI-assisted reactivation yield of denatured GAPDH upon dilution decreases. At 37.0 degrees C, the spontaneous reactivation, PDI-assisted reactivation and intrinsic molar enthalpy change during the PDI-assisted refolding of GAPDH are not detected.     

Chaperone activity of DsbC.

DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.     

Assisted folding of D-glyceraldehyde-3-phosphate dehydrogenase by trigger factor

The Escherichia coli trigger factor is a peptidyl-prolyl cis-trans isomerase that catalyzes proline-limited protein folding extremely well. Here, refolding of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of trigger factor was investigated. The regain of activity of GAPDH was markedly increased by trigger factor after either long- or short-term denaturation, and detectable aggregation of GAPDH intermediates was prevented. In both cases, time courses of refolding of GAPDH were decelerated by trigger factor. The reactivation yield of GAPDH showed a slow down-turn when molar ratios of trigger factor to GAPDH were above 5, due to tight binding between trigger factor and GAPDH intermediates. Such inactive bound GAPDH could be partially rescued from trigger factor by addition of reduced alphaLA as competitor, by further diluting the refolding mixture, or by disrupting hydrophobic interactions in the complexes. A model for trigger factor assisted refolding of GAPDH is proposed. We also suggest that assisted refolding of GAPDH is due mainly to the chaperone function of trigger factor.     

A stable cold folding intermediate of rabbit muscle D-glyceraldehyde 3-phosphate dehydrogenase.

With decreasing temperature the reactivation yield of denatured D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) upon dilution increases but the reactivation rate decreases. Neither reactivation nor aggregation during refolding can be detected at 4 degrees C in 48 h, and at 3 degrees C even in 6 days. However, the reactivation takes place once the temperature is raised with little decrease of the yield after incubation for 6 days at 3 degrees C. A cold folding intermediate forms in a burst phase of refolding at 4 degrees C as shown by a fast change of the intrinsic fluorescence followed by further conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially folded GAPDH subunit with secondary structure between that of the native and denatured enzymes, a hydrophobic cluster not found in either the native or the denatured state, and an active site similar to but different from that of the native state. Chaperonin 60 (GroEL) binds with all intermediates formed at 4 degrees C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustment when dissociated from GroEL in the presence of ATP and further folded and assembled into the native tetramer.     

Dimeric trigger factor stably binds folding-competent intermediates and cooperates with the DnaK-DnaJ-GrpE chaperone system to allow refolding

Trigger factor (TF) is the first chaperone encountered by the nascent chain in bacteria and forms a stoichiometric complex with the ribosome. However, the functional significance of the high cytosolic concentration of uncomplexed TF, the majority of which is dimeric, is unknown. To gain insight into TF function, we investigated the TF concentration dependence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reactivation yield in the presence and absence of the DnaK-DnaJ-GrpE chaperone system in vitro. Cross-linking results indicate that the observed decrease in the reactivation yield of GAPDH at high concentrations of TF is due to the formation of a stable complex between TF dimer and GAPDH intermediates. In the absence of TF, or at low TF concentrations, the DnaK-DnaJ-GrpE chaperone system had negligible effect on the GAPDH refolding yield. However, GAPDH intermediates bound and held by dimeric TF could be specifically rescued by the DnaK-DnaJ-GrpE chaperone system in an ATP-dependent manner. This indicates the potential of TF, in its dimeric form, to act as a binding chaperone, maintaining non-native proteins in a refolding competent conformation and cooperating with downstream molecular chaperones to facilitate post-translational or post-stress protein folding.     

Functional analysis of tunicamycin-inducible gene A polypeptide from Aspergillus niger.

Tunicamycin-inducible gene A polypeptide (TIGA) is a member of the protein disulfide isomerase (PDI) family and is suggested to facilitate the folding of nascent polypeptides. The functional properties of TIGA were investigated here. TIGA acted as an isomerase, catalyzing the refolding of denatured and reduced ribonuclease A. TIGA also exhibited chaperone activity in the refolding of denatured prochymosin but not in the refolding of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), indicating that it had substrate specificity with respect to chaperone activity. Detailed study with a series of thioredoxin-motif (trx-motif) mutants revealed that the 2 trx-motifs of TIGA were not equal in activity. The N-terminal trx-motif was more active than the C-terminal trx-motif, and the first cysteine in each trx-motif was necessary for isomerase activity.     

Interaction of GroE with an all-beta-protein.

Molecular chaperones are involved in protein folding both in vivo and in vitro. The Escherichia coli chaperone GroEL interacts with a number of nonnative proteins. A common structural motif of nonnative proteins, which is recognized by GroEL, has not yet been identified. In order to study the role of beta-sheet secondary structure on the interaction of nonnative proteins with GroEL, we used the F(ab) fragment of a monoclonal antibody as a model substrate protein. Here we show that GroEL interacts functionally with this all-beta-protein during reactivation. Antibody fragments refold spontaneously in good yield from the guanidine-denatured state. Functional refolding to the native state is inhibited transiently by GroEL, but there is no complete folding arrest in the absence of Mg-ATP and GroES. The yield of these unspecifically released GroEL-bound F(ab) fragments corresponds to that of the spontaneous reactivation in the absence of chaperones. However, the refolding kinetics in the presence of GroEL are considerably slower. The addition of Mg-ATP to the GroEL.F(ab) complex results in an immediate release of bound substrate protein and a significant increase in the amount of reconstituted antibody fragments compared to spontaneous reactivation. GroES is not essential for functional GroEL-mediated refolding of the F(ab) fragment but affects the reactivation yield to a small extent. Interestingly, stimulation of the GroEL-mediated F(ab) refolding depends primarily on the binding and not on hydrolysis of adenosine triphosphates. Previous results indicate the binding of alpha-helices to GroEL. The results presented in this paper suggest that beta-sheet secondary structural elements are recognized by GroEL. We therefore conclude that the interaction of a nonnative protein with GroEL depends mainly on the nature of the early folding intermediate but not on a specific element of secondary structure.     

Chaperone discovery

Molecular chaperones assist de novo protein folding and facilitate the refolding of stress‐denatured proteins. The molecular chaperone concept was coined nearly 35 years ago, and since then, tremendous strides have been made in understanding how these factors support protein folding. Here, we focus on how various chaperone proteins were first identified to play roles in protein folding. Examples are used to illustrate traditional routes of chaperone discovery and point out their advantages and limitations. Recent advances, including the development of folding biosensors and promising methods for the stabilization of proteins in vivo, provide new routes for chaperone discovery.     

Effects of macromolecular crowding on the unfolding and the refolding of D-glyceraldehyde-3-phosophospate dehydrogenase

The effects of crowding agents, polyethylene glycol (PEG 20K), Dextran 70, and bovine serum albumin, on the denaturation of homotetrameric D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in 0.5 M guanidine hydrochloride and the reactivation of the fully denatured enzyme have been examined quantitatively. Increasing the concentration of PEG 20K to 225 mg/ml decreases the rate constant of slow phase of GAPDH inactivation to 5% but with no change for the fast phase. Chaperone GroEL assists GAPDH refolding greatly and shows even higher efficiency under crowding condition. Crowding mainly affects refolding steps after the formation of the dimeric folding intermediate.     

Non-functioning chaperonin GroEL stimulates protein aggregation

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to be decreased in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The size of individual proteins (GroEL, GAPDH, and the recombinant prion) and aggregates formed after their mixing have been determined by the dynamic light scattering analysis. It was shown that at 25°C, the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the size of the aggregates to 70–80 nm. After blocking of one of the chaperonin active sites by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.     

Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.     

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.     

The folding of nascent mitochondrial aspartate aminotransferase synthesized in a cell-free extract can be assisted by GroEL and GroES

At 30 degrees C, the precursor to mitochondrial aspartate aminotransferase (pmAspAT) cannot fold after synthesis in rabbit reticulocyte lysate (RRL), a model for studying intracellular protein folding. However, it folds rapidly once imported into mitochondria. Guanidinium chloride denatured pmAspAT likewise cannot refold at 30 degrees C in a defined in vitro system. However, it refolds rapidly and in good yield in the presence of the intramitochondrial chaperone homologues GroEL and GroES. In this report, we demonstrate that GroEL and GroES can also facilitate the folding of nascent pmAspAT in reticulocyte lysate under conditions where it otherwise would not. When added alone, GroEL arrests the slow folding of nascent pmAspAT and inhibits import into mitochondria. These effects are significantly reversed by adding GroES. These observations suggest that added GroEL participates in an equilibrium with endogenous chaperones in the cytosol which inhibit folding and promote import competence. Native gel electrophoresis suggests that nascent pmAspAT exists in RRL as a heterogeneous population of partially folded species, some of which bind to added GroEL more readily than others. The GroEL-trapped species appear to be among the productive pmAspAT folding intermediates formed in RRL or they at least appear to equilibrate with these intermediates, since they become import competent after GroES-stimulated release from GroEL.     

Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase

In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL/GroES-assisted refolding of denatured protein, the yield of correctly folded protein from glycerol- or proline-assisted spontaneous refolding process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance.     

Glycosylation inhibits the interaction of invertase with the chaperone GroEL.

During refolding and reassociation of chemically denatured non-glycosylated invertase from Saccharomyces cerevisiae, aggregation competes with correct folding, leading to low yields of reactivation (Kern et al. (1992) Protein Sci. 1, 120-131). In the presence of the chaperone GroEL, refolding is completely arrested. This suggests the formation of a stable complex between GroEL and non-native non-glycosylated invertase. Addition of MgATP results in a slow release of active invertase from the chaperone complex. When GroEL/ES and MgATP are present during refolding, the final reactivation yield increases from 14% to 36%. In contrast, refolding of the core-glycosylated and the high-mannose glycosylated forms of invertase is not arrested by GroEL. Only a short lag phase at the beginning of reactivation and a slightly increased reactivation yield (64% to 86% for core-glycosylated and 62% to 76% for external invertase) indicate a weak interaction of the glycosylated forms with the chaperone.     

Aggregated proteins accelerate but do not increase the aggregation of D-glyceraldehyde-3-phosphate dehydrogenase. Specificity of protein aggregation

The effect of protein aggregates on the aggregation of d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during unfolding and refolding has been studied. The aggregation of GAPDH follows a sigmoid course. The presence of protein aggregates increases the aggregation rate during unfolding and refolding of GAPDH but does not change the extent of aggregation and the final renaturation yield. It is suggested that protein aggregates function as seeds for aggregation via hydrophobic interaction with only GAPDH folding intermediates destined to aggregate and do not affect the distribution between pathways leading to correct folding and aggregation. Moreover, two different proteins do not interfere with each other during their simultaneous refolding together in a buffer. These findings provide insight into a mechanism by which cells prevent protein folding against the interference from aggregation of other proteins.     

Study on the chaperone properties of conserved GTPases

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.     

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.