Application of a chaperone-based refolding method to two- and three-dimensional off-lattice protein models

A model of protein-chaperone interaction as a two-phase (unfolding/refolding) iterative annealing mechanism able to promote structural segregation of hydrophobic and hydrophilic monomers and thereby facilitate access to nativelike states has recently been applied successfully to two 22-mers of the Honeycutt and Thirumalai BLN (hydrophobic, hydrophilic, neutral) heteropolymer model. This technique is here applied to a much wider data set: 94 8-mers of the off-lattice protein model originally presented in two dimensions by Stillinger and Head-Gordon, and later extended into three dimensions by Irb?ck and Potthast; the model chaperone is shown to be equally successful, and by progressive elaboration of the chaperone model as in the earlier BLN model work, to be utilizing very similar underlying mechanisms. It is demonstrated that on average, contacts with the model chaperone give rise to a consistent movement in structure space in the direction of more nativelike structures; this method of global minimization does not therefore rely fundamentally on random search. Insofar as the responses to the chaperone of the two- and three-dimensional forms of the substrate model do differ, this can be interpreted as reflecting the different handling of hydrophilic monomers in the models-in particular, whether there is active repulsion between these and monomers of hydrophobic character. The chaperone-induced refolding method is also tested on a set of 220 9-mer chains of each version of the substrate model, where it is seen that the two-dimensional model, with its more clearly distinguished roles for the hydrophobic and hydrophilic monomers, shows a more favorable scaling behavior.     

Chaperonin-mediated protein folding: fate of substrate polypeptide

Chaperonins are megadalton ring assemblies that mediate essential ATP-dependent assistance of protein folding to the native state in a variety of cellular compartments, including the mitochondrial matrix, the eukaryotic cytosol, and the bacterial cytoplasm. Structural studies of the bacterial chaperonin, GroEL, both alone and in complex with its co-chaperonin, GroES, have resolved the states of chaperonin that bind and fold non-native polypeptides. Functional studies have resolved the action of ATP binding and hydrolysis in driving the GroEL-GroES machine through its folding-active and binding-active states, respectively. Yet the exact fate of substrate polypeptide during these steps is only poorly understood. For example, while binding involves multivalent interactions between hydrophobic side-chains facing the central cavity of GroEL and exposed hydrophobic surfaces of the non-native protein, the structure of any polypeptide substrate while bound to GroEL remains unknown. It is also unclear whether binding to an open GroEL ring is accompanied by structural changes in the non-native substrate, in particular whether there is an unfolding action. As a polypeptide-bound ring becomes associated with GroES, do the large rigid-body movements of the GroEL apical domains serve as another source of a potential unfolding action? Regarding the encapsulated folding-active state, how does the central cavity itself influence the folding trajectory of a substrate? Finally, how do GroEL and GroES serve, as recently recognized, to assist the folding of substrates too large to be encapsulated inside the machine? Here, such questions are addressed with the findings available to date, and means of further resolving the states of chaperonin-associated polypeptide are discussed.     

Interaction of oxidized chaperonin GroEL with an unfolded protein at low temperatures

The chaperonin GroEL binds to non-native substrate proteins via hydrophobic interactions, preventing their aggregation, which is minimized at low temperatures. In the present study, we investigated the refolding of urea-denatured rhodanese at low temperatures, in the presence of ox-GroEL (oxidized GroEL), which contains increased exposed hydrophobic surfaces and retains its ability to hydrolyse ATP. We found that ox-GroEL could efficiently bind the urea-unfolded rhodanese at 4°C, without requiring excess amount of chaperonin relative to normal GroEL (i.e. non-oxidized). The release/reactivation of rhodanese from GroEL was minimal at 4°C, but was found to be optimal between 22 and 37°C. It was found that the loss of the ATPase activity of ox-GroEL at 4°C prevented the release of rhodanese from the GroEL-rhodanese complex. Thus ox-GroEL has the potential to efficiently trap recombinant or non-native proteins at 4°C and release them at higher temperatures under appropriate conditions.     

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.     

Hydrogen peroxide induces the dissociation of GroEL into monomers that can facilitate the reactivation of oxidatively inactivated rhodanese

Although, several studies have been reported on the effects of oxidants on the structure and function of other molecular chaperones, no reports have been made so far for the chaperonin GroEL. The ability of GroEL to function under oxidative stress was investigated in this report by monitoring the effects of hydrogen peroxide (H(2)O(2)) on the structure and refolding activity of this protein. Using fluorescence spectroscopy and light scattering, we observed that GroEL showed increases in exposed hydrophobic sites and changes in tertiary and quaternary structure. Differential sedimentation, gel electrophoresis, and circular dichroism showed that H(2)O(2) treated GroEL underwent irreversible dissociation into monomers with partial loss of secondary structure. Relative to other proteins, GroEL was found to be highly resistant to oxidative damage. Interestingly, GroEL monomers produced under these conditions can facilitate the reactivation of H(2)O(2)-inactivated rhodanese but not urea-denatured rhodanese. Recovery of approximately 84% active rhodanese was obtained with either native or oxidized GroEL in the absence of GroES or ATP. In comparison, urea-denatured GroEL, BSA and the refolding mixture in the absence of proteins resulted in the recovery of 72, 50, and 49% rhodanese activity, respectively. Previous studies have shown that GroEL monomers can reactivate rhodanese. Here, we show that oxidized monomeric GroEL can reactivate oxidized rhodanese suggesting that GroEL retains the ability to protect proteins during oxidative stress.     

Polyols induce ATP-independent folding of GroEL-bound bacterial glutamine synthetase.

We have previously assessed the GroE chaperonin requirements for folding of bacterial glutamine synthetase (GS) and established that, at 37 degrees C in 50 mM Tris buffer, ATP binding to the GroEL-GS complex is mandatory for the release and reactivation of dodecameric enzyme. However, we demonstrate here that the addition of 1-4 M glycerol to GroEL-GS complexes resulted in release and reactivation of GS in the absence of nucleotide. Furthermore, the kinetics of refolding and refolding yields of this glycerol-induced refolding were similar to those observed with ATP. Other polyols such as sucrose, 1,2-propanediol, or 1,3-propanediol also facilitated nucleotide-independent refolding of GS from chaperonin complex. The observed phenomenon cannot be attributed to the viscosity or molecular crowding effects because solutions of dextran or Ficoll with the same viscosity as 4 M glycerol failed to reactivate GroEL-bound GS. Like glycerol, other osmolytes such as betaine and sarcosine or high salt (500 mM NaCl) facilitated spontaneous folding of GS. However, no reactivation of GroEL-bound GS was observed with these additives. The presence of glycerol affected binding of fluorescent probe 1,8-anilinonaphthalene to GroEL, suggesting that glycerol may alter the chaperonin structure. Our data suggest that low-molecular-weight polyols affect both GroEL and bound GS monomers to reduce their binding affinity. This results in an increased partitioning of GS toward active, assembly-competent states.     

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.     

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.     

Conformational specificity of the chaperonin GroEL for the compact folding intermediates of alpha-lactalbumin.

The chaperonin GroEL binds unfolded polypeptides, preventing aggregation, and then mediates their folding in an ATP-dependent process. To understand the structural features in non-native polypeptides recognized by GroEL, we have used alpha-lactalbumin (alpha LA) as a model substrate. alpha LA (14.2 kDa) is stabilized by four disulfide bonds and a bound Ca2+ ion, offering the possibility of trapping partially folded disulfide intermediates between the native and the fully unfolded state. The conformers of alpha LA with high affinity for GroEL are compact, containing up to three disulfide bonds, and have significant secondary structure, but lack stable tertiary structure and expose hydrophobic surfaces. Complex formation requires almost the complete alpha LA sequence and is strongly dependent on salts that stabilize hydrophobic interactions. Unfolding of alpha LA to an extended state as well as the burial of hydrophobic surface upon formation of ordered tertiary structure prevent the binding to GroEL. Interestingly, GroEL interacts only with a specific subset of the many partially folded disulfide intermediates of alpha LA and thus may influence in vitro the kinetics of the folding pathways that lead to disulfide bonds with native combinations. We conclude that the chaperonin interacts with the hydrophobic surfaces exposed by proteins in a flexible compact intermediate or molten globule state.     

Inter-ring communication allows the GroEL chaperonin complex to distinguish between different substrates

The productive folding of substrate proteins by the GroEL complex of Escherichia coli requires the activity of both the chaperonin rings. These heptameric rings were shown to regulate the chaperonins' affinity for substrates and co-chaperonin via inter-ring communications; however, the molecular details of the interactions are not well understood. We have investigated the effect of substrate binding on inter-ring communications of the chaperonin complex, both the double-ring GroEL as well as the single-ring SR1 chaperonin in complex with four different substrates by using mass spectrometry. This approach shows that whereas SR1 is unable to distinguish between Rubisco, gp23, gp5, and MDH, GroEL shows clear differences upon binding these substrates. The most distinctive binding behavior is observed for Rubisco, which only occupies one GroEL ring. Both bacteriophage capsid proteins (gp23 and gp5) as well as MDH are able to bind to the two GroEL rings simultaneously. Our data suggest that inter-ring communication allows the chaperonin complex to differentiate between substrates. Using collision induced dissociation in the gas phase, differences between the chaperonin(substrate) complexes are observed only when both rings are present. The data indicate that the size of the substrate is an important factor that determines the degree of stabilization of the chaperonin complex.     

Action of the Chaperonin GroEL/ES on a Non-native Substrate Observed with Single-Molecule FRET

The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.     

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.     

Factors governing the substrate recognition by GroEL chaperone: a sequence correlation approach

The chaperonin GroEL binds to a large number of polypeptides, prevents their self-association, and mediates appropriate folding in a GroES and adenosine triphosphate-dependent manner. But how the GroEL molecule actually recognizes the polypeptide and what are the exact GroEL recognition sites in the substrates are still poorly understood. We have examined more than 50 in vivo substrates as well as well-characterized in vitro substrates, for their binding characteristics with GroEL. While addressing the issue, we have been driven by the basic concept that GroES, being the cochaperonin of GroEL, is the best-suited substrate for GroEL, as well as by the fact that polypeptide substrate and GroES occupy the same binding sites on the GroEL apical domain. GroES interacts with GroEL through selective hydrophobic residues present on its mobile loop region, and we have considered the group of residues on the GroES mobile loop as the key element in choosing a substrate for GroEL. Considering the hydrophobic region on the GroES mobile loop as the standard, we have attempted to identify the homologous region on the peptide sequences in the proteins of our interest. Polypeptides have been judged as potential GroEL substrates on the basis of the presence of the GroES mobile loop-like hydrophobic segments in their amino acid sequences. We have observed 1 or more GroES mobile loop-like hydrophobic patches in the peptide sequence of some of the proteins of our interest, and the hydropathy index of most of these patches also seems to be approximately close to that of the standard. It has been proposed that the presence of hydrophobic patches having substantial degree of hydropathy index as compared with the standard segment is a necessary condition for a peptide sequence to be recognized by GroEL molecules. We also observed that the overall hydrophobicity is also close to 30% in these substrates, although this is not the sufficient criterion for a polypeptide to be assigned as a substrate for GroEL. We found that the binding of aconitase, alpha-lactalbumin, and murine dihydrofolate reductase to GroEL falls in line with our present model and have also predicted the exact regions of their binding to GroEL. On the basis of our GroEL substrate prediction, we have presented a model for the binding of apo form of some proteins to GroEL and the eventual formation of the holo form. Our observation also reveals that in most of the cases, the GroES mobile loop-like hydrophobic patch is present in the unstructured region of the protein molecule, specifically in the loop or beta-sheeted region. The outcome of our study would be an essential feature in identifying a potential substrate for GroEL on the basis of the presence of 1 or more GroES mobile loop-like hydrophobic segments in the amino acid sequence of those polypeptides and their location in three-dimensional space.     

Weak Intra-Ring Allosteric Communications of the Archaeal Chaperonin Thermosome Revealed by Normal Mode Analysis

Chaperonins are molecular machines that use ATP-driven cycles to assist misfolded substrate proteins to reach the native state. During the functional cycle, these machines adopt distinct nucleotide-dependent conformational states, which reflect large-scale allosteric changes in individual subunits. Distinct allosteric kinetics has been described for the two chaperonin classes. Bacterial (group I) chaperonins, such as GroEL, undergo concerted subunit motions within each ring, whereas archaeal and eukaryotic chaperonins (group II) undergo sequential subunit motions. We study these distinct mechanisms through a comparative normal mode analysis of monomer and double-ring structures of the archaeal chaperonin thermosome and GroEL. We find that thermosome monomers of each type exhibit common low-frequency behavior of normal modes. The observed distinct higher-frequency modes are attributed to functional specialization of these subunit types. The thermosome double-ring structure has larger contribution from higher-frequency modes, as it is found in the GroEL case. We find that long-range intersubunit correlation of amino-acid pairs is weaker in the thermosome ring than in GroEL. Overall, our results indicate that distinct allosteric behavior of the two chaperonin classes originates from different wiring of individual subunits as well as of the intersubunit communications.     

Exploring the kinetic requirements for enhancement of protein folding rates in the GroEL cavity

The chaperonin system, GroEL and GroES of Escherichia coli enable certain proteins to fold under conditions when spontaneous folding is prohibitively slow as to compete with other non-productive channels such as aggregation. We investigated the plausible mechanisms of GroEL-mediated folding using simple lattice models. In particular, we have investigated protein folding in a confined environment, such as those offered by the GroEL, to decipher whether rate and yield enhancement can occur when the substrate protein is allowed to fold within the cavity of the chaperonins. The GroEL cavity is modeled as a cubic box and a simple bead model is used to represent the substrate chain. We consider three distinct characteristic of the confining environment. First, the cavity is taken to be a passive Anfinsen cage in which the walls merely reduce the available conformation space. We find that at temperatures when the native conformation is stable, the folding rate is retarded in the Anfinsen cage. We then assumed that the interior of the wall is hydrophobic. In this case the folding times exhibit a complex behavior. When the strength of the interaction between the polypeptide chain and the cavity is too strong or too weak we find that the rates of folding are retarded compared to spontaneous folding. There is an optimum range of the interaction strength that enhances the rates. Thus, above this value there is an inverse correlation between the folding rates and the strength of the substrate-cavity interactions. The optimal hydrophobic walls essentially pull the kinetically trapped states which leads to a smoother the energy landscape. It is known that upon addition of ATP and GroES the interior cavity of GroEL offers a hydrophilic-like environment to the substrate protein. In order to mimic this within the context of the dynamic Anfinsen cage model, we allow for changes in the hydrophobicity of the walls of the cavity. The duration for which the walls remain hydrophobic during one cycle of ATP hydrolysis is allowed to vary. These calculations show that frequent cycling of the wall hydrophobicity can dramatically reduce the folding times and increase the yield as well under non-permissive conditions. Examination of the structures of the substrate proteins before and after the change in hydrophobicity indicates that there is global unfolding involved. In addition, it is found that a fraction of the molecules kinetically partition to the native state in accordabce with the iterative annealing mechanism. Thus, frequent "unfoldase" activity of chaperonins leading to global unfolding of the polypeptide chain results in enhancement of the folding rates and yield of the folded protein. We suggest that chaperonin efficiency can be greatly enhanced if the cycling time is reduced. The calculations are used to interpret a few experiments on chaperonin-mediated protein folding.     

Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.     

A thermodynamic coupling mechanism can explain the GroEL-mediated acceleration of the folding of barstar

Despite extensive structural and kinetic studies, the mechanism by which the Escherichia coli chaperonin GroEL assists protein folding has remained somewhat elusive. It appears that GroEL might play an active role in facilitating folding, in addition to its role in restricting protein aggregation by secluding folding intermediates. We have investigated the kinetic mechanism of GroEL-mediated refolding of the small protein barstar. GroEL accelerates the observed fast (millisecond) refolding rate, but it does not affect the slow refolding kinetics. A thermodynamic coupling mechanism, in which the concentration of exchange-competent states is increased by the law of mass action, can explain the enhancement of the fast refolding rates. It is not necessary to invoke a catalytic role for GroEL, whereby either the intrinsic refolding rate of a productive folding transition or the unfolding rate of a kinetically trapped off-pathway intermediate is increased by the chaperonin.     

Multi-scale simulations provide supporting evidence for the hypothesis of intramolecular protein translocation in GroEL/GroES complexes

The biological function of chaperone complexes is to assist the folding of non-native proteins. The widely studied GroEL chaperonin is a double-barreled complex that can trap non-native proteins in one of its two barrels. The ATP-driven binding of a GroES cap then results in a major structural change of the chamber where the substrate is trapped and initiates a refolding attempt. The two barrels operate anti-synchronously. The central region between the two barrels contains a high concentration of disordered protein chains, the role of which was thus far unclear. In this work we report a combination of atomistic and coarse-grained simulations that probe the structure and dynamics of the equatorial region of the GroEL/GroES chaperonin complex. Surprisingly, our simulations show that the equatorial region provides a translocation channel that will block the passage of folded proteins but allows the passage of secondary units with the diameter of an alpha-helix. We compute the free-energy barrier that has to be overcome during translocation and find that it can easily be crossed under the influence of thermal fluctuations. Hence, strongly non-native proteins can be squeezed like toothpaste from one barrel to the next where they will refold. Proteins that are already fairly close to the native state will not translocate but can refold in the chamber where they were trapped. Several experimental results are compatible with this scenario, and in the case of the experiments of Martin and Hartl, intra chaperonin translocation could explain why under physiological crowding conditions the chaperonin does not release the substrate protein.     

Polypeptide in the chaperonin cage partly protrudes out and then folds inside or escapes outside

The current mechanistic model of chaperonin-assisted protein folding assumes that the substrate protein in the cage, formed by GroEL central cavity capped with GroES, is isolated from outside and exists as a free polypeptide. However, using ATPase-deficient GroEL mutants that keep GroES bound, we found that, in the rate-limiting intermediate of a chaperonin reaction, the unfolded polypeptide in the cage partly protrudes through a narrow space near the GroEL/GroES interface. Then, the entire polypeptide is released either into the cage or to the outside medium. The former adopts a native structure very rapidly and the latter undergoes spontaneous folding. Partition of the in-cage folding and the escape varies among substrate proteins and is affected by hydrophobic interaction between the polypeptide and GroEL cavity wall. The ATPase-active GroEL with decreased in-cage folding produced less of a native model substrate protein in Escherichia coli cells. Thus, the polypeptide in the critical GroEL-GroES complex is neither free nor completely confined in the cage, but it is interacting with GroEL's apical region, partly protruding to outside.     

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.