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

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

Structural plasticity and noncovalent substrate binding in the GroEL apical domain. A study using electrospay ionization mass spectrometry and fluorescence binding studies

Advances in understanding how GroEL binds to non-native proteins are reported. Conformational flexibility in the GroEL apical domain, which could account for the variety of substrates that GroEL binds, is illustrated by comparison of several independent crystallographic structures of apical domain constructs that show conformational plasticity in helices H and I. Additionally, ESI-MS indicates that apical domain constructs have co-populated conformations at neutral pH. To assess the ability of different apical domain conformers to bind co-chaperone and substrate, model peptides corresponding to the mobile loop of GroES and to helix D from rhodanese were studied. Analysis of apical domain-peptide complexes by ESI-MS indicates that only the folded or partially folded apical domain conformations form complexes that survive gas phase conditions. Fluorescence binding studies show that the apical domain can fully bind both peptides independently. No competition for binding was observed, suggesting the peptides have distinct apical domain-binding sites. Blocking the GroES-apical domain-binding site in GroEL rendered the chaperonin inactive in binding GroES and in assisting the folding of denatured rhodanese, but still capable of binding non-native proteins, supporting the conclusion that GroES and substrate proteins have, at least partially, distinct binding sites even in the intact GroEL tetradecamer.     

Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.     

Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.     

GroEL-substrate-GroES ternary complexes are an important transient intermediate of the chaperonin cycle

GroEL C138W is a mutant form of Escherichia coli GroEL, which forms an arrested ternary complex composed of GroEL, the co-chaperonin GroES and the refolding protein molecule rhodanese at 25 degrees C. This state of arrest could be reversed with a simple increase in temperature. In this study, we found that GroEL C138W formed both stable trans- and cis-ternary complexes with a number of refolding proteins in addition to bovine rhodanese. These complexes could be reactivated by a temperature shift to obtain active refolded protein. The simultaneous binding of GroES and substrate to the cis ring suggested that an efficient transfer of substrate protein into the GroEL central cavity was assured by the binding of GroES prior to complete substrate release from the apical domain. Stopped-flow fluorescence spectroscopy of the mutant chaperonin revealed a temperature-dependent conformational change in GroEL C138W that acts as a trigger for complete protein release. The behavior of GroEL C138W was reflected closely in its in vivo characteristics, demonstrating the importance of this conformational change to the overall activity of GroEL.     

Rhodanese can partially refold in its GroEL-GroES-ADP complex and can be released to give a homogeneous product

Molecular chaperones GroEL and GroES facilitate reactivation of denatured rhodanese which folds poorly unless the process is assisted. The present work tests the hypothesis that more extensively unfolded forms of rhodanese bind tighter than those forms that appear later in the folding pathway. The study of the interaction of different urea-induced forms of rhodanese with GroEL suggests that species preceding the domain folded form bind directly and productively to GroEL. Rhodanese partially folds while in the GroEL-GroES-ADP complex, but it does not significantly reach an active state. Partially folded rhodanese can be released from the GroEL-GroES-ADP complex by subdenaturing concentrations of urea as a homogeneous species that is committed to fold to the native conformation with little or no partitioning to the aggregated state. Dilution of denatured rhodanese to the same final concentration gives less active enzyme and significant aggregation. Urea denaturation studies show that active rhodanese released from complexes behaves identically to native enzyme, while spontaneously folded rhodanese has a different stability. These results are interpreted using a previously proposed model based on studies of unassisted rhodanese folding [Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta 1382, 120-128. Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63-70].     

Analysis of peptides and proteins in their binding to GroEL

The GroEL–GroES is an essential molecular chaperon system that assists protein folding in cell. Binding of various substrate proteins to GroEL is one of the key aspects in GroEL‐assisted protein folding. Small peptides may mimic segments of the substrate proteins in contact with GroEL and allow detailed structural analysis of the interactions. A model peptide SBP has been shown to bind to a region in GroEL that is important for binding of substrate proteins. Here, we investigated whether the observed GroEL–SBP interaction represented those of GroEL–substrate proteins, and whether SBP was able to mimic various aspects of substrate proteins in GroE‐assisted protein folding cycle. We found that SBP competed with substrate proteins, including α‐lactalbumin, rhodanese, and malate dehydrogenase, in binding to GroEL. SBP stimulated GroEL ATP hydrolysis rate in a manner similar to that of α‐lactalbumin. SBP did not prevent GroES from binding to GroEL, and GroES association reduced the ATPase rates of GroEL/SBP and GroEL/α‐lactalbumin to a comparable extent. Binding of both SBP and α‐lactalbumin to apo GroEL was dominated by hydrophobic interaction. Interestingly, association of α‐lactalbumin to GroEL/GroES was thermodynamically distinct from that to GroEL with reduced affinity and decreased contribution from hydrophobic interaction. However, SBP did not display such differential binding behaviors to apo GroEL and GroEL/GroES, likely due to the lack of a contiguous polypeptide chain that links all of the bound peptide fragments. Nevertheless, studies using peptides provide valuable information on the nature of GroEL–substrate protein interaction, which is central to understand the mechanism of GroEL‐assisted protein folding. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Determination of the number of active GroES subunits in the fused heptamer GroES required for interactions with GroEL

A double-heptamer ring chaperonin GroEL binds denatured substrate protein, ATP, and GroES to the same heptamer ring and encapsulates substrate into the central cavity underneath GroES where productive folding occurs. GroES is a disk-shaped heptamer, and each subunit has a GroEL-binding loop. The residues of the GroEL subunit responsible for GroES binding largely overlap those involved in substrate binding, and the mechanism by which GroES can replace the substrate when GroES binds to GroEL/substrate complex remains to be clarified. To address this question, we generated single polypeptide GroES by fusing seven subunits with various combinations of active and GroEL binding-defective subunits. Functional tests of the fused GroES variants indicated that four active GroES subunits were required for efficient formation of the stable GroEL/GroES complex and five subunits were required for the productive GroEL/substrate/GroES complex. An increase in the number of defective GroES subunits resulted in a slowing of encapsulation and folding. These results indicate the presence of an intermediate GroEL/substrate/GroES complex in which the substrate and GroES bind to GroEL by sharing seven common binding sites.     

The lower hydrolysis of ATP by the stress protein GroEL is a major factor responsible for the diminished chaperonin activity at low temperature

The chaperonins GroEL and GroES were shown to facilitate the refolding of urea-unfolded rhodanese in an ATP-dependent process at 25 or 37 degrees C. A diminished chaperonin activity was observed at 10 degrees C, however. At low temperature, GroEL retains its ability to form a complex with urea-unfolded rhodanese or with GroES. GroEL is also able to bind ATP at 10 degrees C. Interestingly, the ATPase activity of GroEL was highly decreased at low temperatures. Hydrolysis of ATP by GroEL was 60% less at 10 degrees C than at 25 degrees C. We conclude that the reduced hydrolysis of ATP by GroEL is a major but perhaps not the only factor responsible for the diminished chaperonin activity at 10 degrees C. GroEL may function primarily at higher temperatures in which the ability of GroEL to hydrolyze ATP is not compromised.     

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.     

GroEL interacts transiently with oxidatively inactivated rhodanese facilitating its reactivation

When the enzyme rhodanese was inactivated with hydrogen peroxide (H(2)O(2)), it underwent significant conformational changes, leading to an increased exposure of hydrophobic surfaces. Thus, this protein seemed to be an ideal substrate for GroEL, since GroEL uses hydrophobic interactions to bind to its substrate polypeptides. Here, we report on the facilitated reactivation (86%) of H(2)O(2)-inactivated rhodanese by GroEL alone. Reactivation by GroEL required a reductant and the enzyme substrate, but not GroES or ATP. Further, we found that GroEL interacted weakly and/or transiently with H(2)O(2)-inactivated rhodanese. A strong interaction with rhodanese was obtained when the enzyme was pre-incubated with urea, indicating that exposure of hydrophobic surfaces alone on oxidized rhodanese was not sufficient for the formation of a strong complex and that a more unfolded structure of rhodanese was required to interact strongly with GroEL. Unlike prior studies that involved denaturation of rhodanese through chemical or thermal means, we have clearly shown that GroEL can function as a molecular chaperone in the reactivation of an oxidatively inactivated protein. Additionally, the mechanism for the GroEL-facilitated reactivation of rhodanese shown here appears to be different than that for the chaperonin-assisted folding of chemically unfolded polypeptides in which a nucleotide and sometimes GroES is required.     

Molecular chaperones: Inside and outside the Anfinsen cage

The GroEL/GroES chaperonin system acts as a passive anti-aggregation cage for refolding rubisco and rhodanese, and not as an active unfolding device. Refolding aconitase is too large to enter the cage but reversible binding to GroEL reduces its aggregration. Unexpectedly, confinement in the cage increases the rate of refolding of rubisco, but not rhodanese.     

Three GroEL homologues from Rhizobium leguminosarum have distinct in vitro properties

The GroEL molecular chaperone of Escherichia coli and its cofactor GroES are highly conserved, and are required for the folding of many proteins. Most but not all bacteria express single GroEL and GroES proteins. Rhizobium leguminosarum strain A34 encodes three complete operons encoding homologues to GroEL and GroES. We have used circular dichroism and measurement of ATPase activity to compare the stabilities of these chaperonins after expression in and purification from E. coli. Significant differences in the stabilities of the proteins with respect to denaturant and temperature were found. The proteins also differed in their ability to refold denatured lactate dehydrogenase. This study, the first to compare the properties of three different GroEL homologues from the same organism, shows that despite the high degree of similarity between different homologues, they can display distinct properties in vitro.     

Role of the gamma-phosphate of ATP in triggering protein folding by GroEL-GroES: function, structure and energetics

Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the gamma-phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already-formed, folding-inactive cis ADP ternary complexes, essentially introducing the gamma-phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (-46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL-GroES-ADP.AlF3 was determined at 2.8 A. A trigonal AlF3 metal complex was observed in the gamma-phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL-GroES-ADP complex, no other differences were observed. We discuss the likely basis of the ability of gamma-phosphate binding to convert preformed GroEL-GroES-ADP-polypeptide complexes into the folding-active state.     

The unfolding action of GroEL on a protein substrate

A molecular dynamics simulation of the active unfolding of denatured rhodanese by the chaperone GroEL is presented. The compact denatured protein is bound initially to the cis cavity and forms stable contacts with several of the subunits. As the cis ring apical domains of GroEL undergo the transition from the closed to the more open (ATP-bound) state, they exert a force on rhodanese that leads to the increased unfolding of certain loops. The contacts between GroEL and rhodanese are analyzed and their variation during the GroEL transition is shown. The major contacts, which give rise to the stretching force, are found to be similar to those observed in crystal structures of peptides bound to the apical domains. The results of the simulation show that multidomain interactions play an essential role, in accord with experiments. Implications of the results for mutation experiments and for the action of GroEL are discussed.     

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.     

GroEL-assisted refolding of adrenodoxin during chemical cluster insertion.

Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP. The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possible scaffolding role for two bacterial sulfur transferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates. The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH-cytochrome c reduction assay. These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin-containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times. The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanese, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein.     

Discrimination of ATP,ADP, and AMPPNP by chaperonin GroEL: hexokinase treatment revealed the exclusive role of ATP

The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident.     

Single molecular observation of the interaction of GroEL with substrate proteins.

To understand the mechanism of GroEL-assisted protein folding, we observed the interaction of fluorescence-labeled GroEL with fluorescence-labeled substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. GroEL with a A133C mutation in the equatorial domain was labeled with a fluorescent dye, tetramethylrhodamine. As substrate proteins, we used the largely denatured and partly denatured forms of bovine beta-lactoglobulin, both labeled with another fluorescent dye, Cy5. The complexes formed by GroEL with these substrates were characterized by size-exclusion gel chromatography. The recovered complexes were then observed by fluorescence microscopy. For both substrates, agreement of the fluorescent spots for tetramethylrhodamine and Cy5 indicated formation of the complex at the single molecule level. Similar observation of macroscopic binding by size-exclusion chromatography and microscopic binding by the fluorescence microscopy was done for the folding intermediate of Cy5-labeled bovine rhodanese. The fluorescence microscopy opens a new avenue for studying the interaction of GroEL with substrate proteins.