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.     

Dual function of protein confinement in chaperonin-assisted protein folding.

The GroEL/GroES chaperonin system mediates the folding of a range of newly synthesized polypeptides in the bacterial cytosol. Using a rapid biotin-streptavidin-based inhibition of chaperonin function, we show that the cage formed by GroEL and its cofactor GroES can have a dual role in promoting folding. First, enclosure of nonnative protein in the GroEL:GroES complex is essential for folding to proceed unimpaired by aggregation. Second, folding inside the cage can be significantly faster than folding in free solution, independently of ATP-driven cycles of GroES binding and release. This suggests that confinement of unfolded protein in the narrow hydrophilic space of the chaperonin cage smoothes the energy landscape for the folding of some proteins, increasing the flux of folding intermediates toward the native state.     

Football- and bullet-shaped GroEL-GroES complexes coexist during the reaction cycle

GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.     

Interaction of GroEL and GroEL/GroES complexes with a nonnative subtilisin variant: a small-angle neutron scattering study

Small-angle neutron scattering and contrast variation were used to study the solution structure of GroEL and GroEL/GroES chaperonins complexed with a nonnative variant of the polypeptide substrate, subtilisin (PJ9). The subtilisin was 86% deuterated (dPJ9) so that it contrasted sufficiently with the chaperonin, allowing the contrast variation technique to be used to separate the scattering from the two components bound in the complex. Both the native double-ring GroEL and a single-ring mutant were used with dPJ9 bound in a 1:1 stoichiometry per GroEL toroid. This allowed both the position and the shape of dPJ9 in the GroEL/dPJ9 complexes to be determined. A single-ring GroEL/GroES variant complexed with one dPJ9 molecule was used to study the structural changes of dPJ9 in GroEL/GroES/dPJ9 complexes formed with ADP and with ATP. It was found that both the shape and the position of the bound dPJ9 in the GroEL/GroES/dPJ9 complex with ADP were the same as those in the GroEL/dPJ9 complex. However, dPJ9 assumed a more symmetric shape when bound in the GroEL/GroES/dPJ9 complex with ATP. This important observation reflects the relative ability of ATP to promote refolding of protein substrates relative to that of ADP.     

GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.     

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.     

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.     

GroEL mediates protein folding with a two successive timer mechanism

GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.     

"Half of the sites" binding of D-glyceraldehyde-3-phosphate dehydrogenase folding intermediate with GroEL

Two D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) folding intermediate subunits bind with chaperonin 60 (GroEL) to form a stable complex, which can no longer bind with additional GAPDH intermediate subunits, but does bind with one more lysozyme folding intermediate or one chaperonin 10 (GroES) molecule, suggesting that the two GAPDH subunits bind at one end of the GroEL molecule displaying a "half of the sites" binding profile. For lysozyme, GroEL binds with either one or two folding intermediates to form a stable 1:1 or 1:2 complex with one substrate on each end of the GroEL double ring for the latter. The 1:1 complex of GroEL.GroES binds with one lysozyme or one dimeric GAPDH folding intermediate to form a stable ternary complex. Both complexes of GroEL.lysozyme1 and GroEL.GAPDH2 bind with one GroES molecule only at the other end of the GroEL molecule forming a trans ternary complex. According to the stoichiometry of GroEL binding with the GAPDH folding intermediate and the formation of ternary complexes containing GroEL.GAPDH2, it is suggested that the folding intermediate of GAPDH binds, very likely in the dimeric form, with GroEL at one end only.     

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.     

A newly synthesized protein interacts with GroES on the surface of chaperonin GroEL.

To facilitate folding and assembly of different proteins, chaperonin GroEL requires the presence of its helper protein GroES. Using a photochemical cross-linking approach, we show that GroES and newly synthesized pre-beta-lactamase (pre-beta lac) contact with each other only within the ternary complex with GroEL. Possibly owing to this contact GroES is able to directly influence the pre-beta lac/GroEL interaction. Furthermore, the cross-linking of pre-beta lac to GroES suggests that the binding of the protein ligands to GroEL occurs near the GroES binding site, known to be in the central hole space of GroEL.     

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.     

Co-expression of chaperonin GroEL/GroES enhances in vivo folding of yeast mitochondrial aconitase and alters the growth characteristics of Escherichia coli

Over last two decades many researchers have demonstrated the mechanisms of how the Escherichia coli chaperonin GroEL and GroES work in the binding and folding of different aggregation prone substrate proteins both in vivo and in vitro. However, preliminary aspects, such as influence of co-expressing GroEL and GroES on the over expression of other recombinant proteins in E. coli cells and subsequent growth aspects, as well as the conditions for optimum production of recombinant proteins in presence of recombinant chaperones have not been properly investigated. In the present study we have demonstrated the temperature dependent growth characteristics of E. coli cells, which are over expressing recombinant aconitase and how the co-expression of E. coli chaperonin GroEL and GroES influence the growth rate of the cells and in vivo folding of recombinant aconitase. Presence of co-expressed GroEL reduces the aconitase over-expression drastically; however, exogenous GroEL & GroES together compensate this reduction. For the aconitase over-expressing cells the growth rate decreases by 30% at 25 degrees C when compared with the M15 E. coli cells, however, there is an increase of 20% at 37 degrees C indicating the participation of endogenous chaperonin in the folding of a fraction of over expressed aconitase. However, in presence of co-expressed GroEL and GroES the growth rate of aconitase producing cells was enhanced by 30% at 37 degrees C confirming the assistance of exogenous chaperone system for the folding of recombinant aconitase. Optimum in vivo folding of aconitase requires co-production of complete E. coli chaperonin machinery GroEL and GroES together.     

A mutation in GroEL interferes with protein folding by reducing the rate of discharge of sequestered polypeptides.

GroEL140, a mutant Escherichia coli chaperonin unable to support bacteriophage lambda head assembly, was purified to near homogeneity and compared to wild type GroEL (cpn60). GroEL140 exhibited a 1.5-fold lower ATPase activity relative to the wild type protein. The hydrolysis of ATP by both polypeptides was fully inhibited by an excess of ATP gamma S and partially inhibited by ADP and 5'-adenylylimidodiphosphate, suggesting that adenine nucleotides display different affinities for the ATP binding site of chaperonins. GroEL140 was more sensitive to trypsin digestion compared to wild type GroEL indicating that the mutation destabilized the conformation of the mutant. The proteolytic susceptibility of both chaperonins was similarly enhanced upon addition of ATP, ADP or non-hydrolyzable ATP analogs, providing evidence (i) of a conformational change in the chaperonin structure which is likely to drive the protein discharge process, and (ii) that hydrolysis of ATP is not required to achieve topological modifications. GroEL140 retained its ability to bind non-native ribulose bisphosphate carboxylase/oxygenase (Rbu-P2-carboxylase), but released bound proteins upon addition of ATP and GroES (cpn 10) 6-7-fold less efficiently compared to GroEL. This functional defect was shown to be related to a suboptimal, but not an absence of, interaction with GroES since (i) GroEL140 and GroES were unable to form a complex isolatable by size exclusion chromatography, and (ii) increasing the incubation time or the concentration of GroES enhanced the amount of refolded Rbu-P2-carboxylase discharged from GroEL140-Rbu-P2-carboxylase binary complexes. Pulse-chase experiments involving a double immunoabsorption technique confirmed that Rbu-P2-carboxylase remained associated two times longer with GroEL140 than with GroEL in vivo.     

BeF(x) stops the chaperonin cycle of GroEL-GroES and generates a complex with double folding chambers

Coupling with ATP hydrolysis and cooperating with GroES, the double ring chaperonin GroEL assists the folding of other proteins. Here we report novel GroEL-GroES complexes formed in fluoroberyllate (BeF(x)) that can mimic the phosphate part of the enzyme-bound nucleotides. In ATP, BeF(x) stops the functional turnover of GroEL by preventing GroES release and produces a symmetric 1:2 GroEL-GroES complex in which both GroEL rings contain ADP.BeF(x) and an encapsulated substrate protein. In ADP, the substrate protein-loaded GroEL cannot bind GroES. In ADP plus BeF(x), however, it can bind GroES to form a stable 1:1 GroEL-GroES complex in which one of GroEL rings contains ADP.BeF(x) and an encapsulated substrate protein. This 1:1 GroEL-GroES complex is converted into the symmetric 1:2 GroEL-GroES complex when GroES is supplied in ATP plus BeF(x). Thus, BeF(x) stabilizes two GroEL-GroES complexes; one with a single folding chamber and the other with double folding chambers. These results shed light on the intermediate ADP.P(i) nucleotide states in the functional cycle of GroEL.     

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.     

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.     

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.     

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.     

An expanded protein folding cage in the GroEL-gp31 complex

Bacteriophage T4 produces a GroES analogue, gp31, which cooperates with the Escherichia coli GroEL to fold its major coat protein gp23. We have used cryo-electron microscopy and image processing to obtain three-dimensional structures of the E.coli chaperonin GroEL complexed with gp31, in the presence of both ATP and ADP. The GroEL-gp31-ADP map has a resolution of 8.2 A, which allows accurate fitting of the GroEL and gp31 crystal structures. Comparison of this fitted structure with that of the GroEL-GroES-ADP structure previously determined by cryo-electron microscopy shows that the folding cage is expanded. The enlarged volume for folding is consistent with the size of the bacteriophage coat protein gp23, which is the major substrate of GroEL-gp31 chaperonin complex. At 56 kDa, gp23 is close to the maximum size limit of a polypeptide that is thought to fit inside the GroEL-GroES folding cage.