The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction: humans versus Escherichia coli

Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a beta-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the beta-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.     

On the oligomeric state of chloroplast chaperonin 10 and chaperonin 20

Type I chaperonins are fundamental protein folding machineries that function in eubacteria, mitochondria and chloroplasts. Eubacteria and mitochondria contain chaperonin systems comprised of homo-oligomeric chaperonin 60 tetradecamers and co-chaperonin 10 heptamers. In contrast, the chloroplast chaperonins are heterooligomeric tetradecamers that are composed of two subunit types, alpha and beta. Additionally, chloroplasts contain two structurally distinct co-chaperonins. One, ch-cpn10, is probably similar to the mitochondrial and bacterial co-chaperonins, and is composed of 10 kDa subunits. The other, termed ch-cpn20 is composed of two cpn10-like domains that are held together by a short linker. While the oligomeric structure of ch-cpn10 remains to be elucidated, it was previously suggested that ch-cpn20 forms tetramers in solution, and that this is the functional oligomer. In the present study, we investigated the properties of purified ch-cpn10 and ch-cpn20. Using bifunctional cross-linking reagents, gel filtration chromatography and analytical ultracentrifugation, we show that ch-cpn10 is a heptamer in solution. In contrast, ch-cpn20 forms multiple oligomers that are in dynamic equilibrium with each other and cover a broad spectrum of molecular weights in a concentration-dependent manner. However, upon association with GroEL, only one type of co-chaperonin-GroEL complex is formed.     

Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria

Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E. coli GroEL. Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin. To define the in vivo role of the co- chaperonin, we have used the genetic and biochemical potential of the yeast S. cerevisiae. The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated. Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function. Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space. The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro. The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature.     

GroEL-mediated protein folding: making the impossible, possible

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins-constrained by sequence, topology, size, and function-simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

GroEL-Mediated Protein Folding: Making the Impossible,Possible

ABSTRACT

Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.     

A mobile loop order-disorder transition modulates the speed of chaperonin cycling

Molecular machines order and disorder polypeptides as they form and dissolve large intermolecular interfaces, but the biological significance of coupled ordering and binding has been established in few, if any, macromolecular systems. The ordering and binding of GroES co-chaperonin mobile loops accompany an ATP-dependent conformational change in the GroEL chaperonin that promotes client protein folding. Following ATP hydrolysis, disordering of the mobile loops accompanies co-chaperonin dissociation, reversal of the GroEL conformational change, and release of the client protein. "High-affinity" GroEL mutants were identified by their compatibility with "low-affinity" co-chaperonin mutants and incompatibility with high-affinity co-chaperonin mutants. Analysis of binding kinetics using the intrinsic fluorescence of tryptophan-containing co-chaperonin variants revealed that excessive affinity causes the chaperonin to stall in a conformation that forms in the presence of ATP. Destabilizing the beta-hairpins formed by the mobile loops restores the normal rate of dissociation. Thus, the free energy of mobile-loop ordering and disordering acts like the inertia of an engine's flywheel by modulating the speed of chaperonin conformational changes.     

Chloroplast �� chaperonins from A. thaliana function with endogenous cpn10 homologs in vitro

The involvement of type I chaperonins in bacterial and organellar protein folding has been well-documented. In E. coli and mitochondria, these ubiquitous and highly conserved proteins form chaperonin oligomers of identical 60 kDa subunits (cpn60), while in chloroplasts, two distinct cpn60 α and β subunit types co-exist together. The primary sequence of α and β subunits is ~50% identical, similar to their respective homologies to the bacterial GroEL. Moreover, the A. thaliana genome contains two α and four β genes. The functional significance of this variability in plant chaperonin proteins has not yet been elucidated. In order to gain insight into the functional variety of the chloroplast chaperonin family members, we reconstituted β homo-oligomers from A. thaliana following their expression in bacteria and subjected them to a structure-function analysis. Our results show for the first time, that A. thaliana β homo-oligomers can function in vitro with authentic chloroplast co-chaperonins (ch-cpn10 and ch-cpn20). We also show that oligomers made up of different β subunit types have unique properties and different preferences for co-chaperonin partners. We propose that chloroplasts may contain active β homo-oligomers in addition to hetero-oligomers, possibly reflecting a variety of cellular roles.     

The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin.     

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the co-chaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, MitCHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Mandel, H. (2008) Am. J. Hum. Genet. 83, 30–42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.Type I chaperonins are essential molecular chaperones of the Hsp60 family found in eubacteria, mitochondria, and chloroplasts (1). They are key players in mediating the correct folding of newly translated, translocated, and stress-denatured proteins. The folding function of chaperonins is executed by the coordinated action of two oligomeric proteins, Hsp60 (also named cpn60 and in bacteria GroEL) and its co-chaperonin Hsp10 (also named cpn10 and in bacteria GroES). The GroEL molecule is composed of 14 subunits that form a barrel-like structure that consists of two back-to-back stacked heptameric rings with a large cavity at each end termed the “Anfinsen cage.” GroES is a heptameric ring formed by ∼10-kDa subunits. The co-chaperonin binds to the chaperonin in the presence of ATP and Mg²⁺ via a short, unstructured, but highly conserved region known as the mobile loop (2, 3). Due to the stability of the GroEL/ES oligomers and the ease with which they can be purified from bacteria, they have become the primary targets for study in the field of chaperonins. As a result, almost all of our knowledge concerning the structure and mechanism of chaperonins, both Hsp60 and Hsp10, is based largely on data from experiments on these E. coli proteins. The chaperonin reaction cycle starts when a non-folded protein substrate binds to the surface of the cavity of one of the GroEL rings. ATP-dependent GroES binding to that (cis) ring causes a dramatic conformational change that leads to a doubling of the cavity size and a switch in the cavity surface from being hydrophobic to hydrophilic. These conformational changes trigger the release of the encapsulated substrate protein into the cavity, where it can fold in a protected environment (4–9). Subsequent binding of ATP and GroES to the opposite (trans) ring promotes the release of GroES, ADP, and protein substrate from the cis ring into bulk solution.The homologous mitochondrial chaperonin system was shown to be responsible for refolding proteins imported into the mitochondria (10, 11). The mitochondrial Hsp60 (mHsp60)³ is similar to GroEL in that it is made up of heptameric rings (12–14) that can refold denatured substrates in vitro with the assistance of a co-chaperonin and ATP (15). However, the mHsp60 oligomer is less stable than the bacterial homolog, and it exhibits unique nucleotide binding properties and specificity for co-chaperonin (13, 14, 16).In addition to their essential function in mediating protein folding, the mammalian mitochondrial chaperonins were also suggested to be involved in extramitochondrial activities. A number of reports have suggested that mHsp60 can stimulate human leukocytes and vascular endothelial cells to produce proinflammatory cytokines (17). Furthermore, it has been reported that mHsp60 has proapoptotic and antiapoptotic roles, depending on its cellular localization (18, 19). Finally, mHsp60 and mHsp10 were found to change their expression pattern in tumor cells (20, 21).The importance of mHsp60 for human cell function has been demonstrated through the autosomal dominant hereditary spastic paraplegia SPG13, a neurodegenerative disorder associated with two independent mutations in the gene encoding mHsp60 (22, 23). Recently, a large kindred including 23 patients suffering from MitCHAP-60 disease, an autosomal recessive neurodegenerative disorder, has been identified (24). Magnetic resonance imaging of the brains of the patients showed diffuse hypomyelination and leukodystrophy, in which myelin is not formed properly. The disease-causing mutation was identified to be a homozygous missense mutation in the human HSPD1 gene encoding the mHsp60 protein (24), namely D3G in the mature protein. Initial studies showed that, in contrast to wild-type mHsp60, the mutant, together with mHsp10, was not able to fully complement a deletion of the bacterial homologues, GroEL and GroES, in E. coli (24). The mechanism by which the D3G mutation may compromise the function of mHsp60 has not been reported. In this study, we suggest that the D3G mutation impedes the function of mHsp60 by entropic destabilization of the oligomeric structure of the molecule.     

Mapping pathways of allosteric communication in GroEL by analysis of correlated mutations

An interesting example of an allosteric protein is the chaperonin GroEL. It undergoes adenosine 5'-triphosphate-induced conformational changes that are reflected in binding of adenosine 5'-triphosphate with positive cooperativity within rings and negative cooperativity between rings. Herein, correlated mutations in chaperonins are analyzed to unravel routes of allosteric communication in GroEL and in its complex with its co-chaperonin GroES. It is shown that analysis of correlated mutations in the chaperonin family can provide information about pathways of allosteric communication within GroEL and between GroEL and GroES. The results are discussed in the context of available structural, genetic, and biochemical data concerning short- and long-range interactions in the GroE system.     

Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin.

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.     

Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.     

Differential effects of co-chaperonin homologs on cpn60 oligomers

In this study, we have investigated the relationship between chaperonin/co-chaperonin binding, ATP hydrolysis, and protein refolding in heterologous chaperonin systems from bacteria, chloroplast, and mitochondria. We characterized two types of chloroplast cpn60 oligomers, ch-cpn60 composed of α and β subunits (α₇β₇ ch-cpn60) and one composed of all β subunits (β₁₄ ch-cpn60). In terms of ATPase activity, the rate of ATP hydrolysis increased with protein concentration up to 60 μM, reflecting a concentration at which the oligomers are stable. At high concentrations of cpn60, all cpn10 homologs inhibited ATPase activity of α₇β₇ ch-cpn60. In contrast, ATPase of β₁₄ ch-cpn60 was inhibited only by mitochondrial cpn10, supporting previous reports showing that β₁₄ is functional only with mitochondrial cpn10 and not with other cpn10 homologs. Surprisingly, direct binding assays showed that both ch-cpn60 oligomer types bind to bacterial, mitochondrial, and chloroplast cpn10 homologs with an equal apparent affinity. Moreover, mitochondrial cpn60 binds chloroplast cpn20 with which it is not able to refold denatured proteins. Protein refolding experiments showed that in such instances, the bound protein is released in a conformation that is not able to refold. The presence of glycerol, or subsequent addition of mitochondrial cpn10, allows us to recover enzymatic activity of the substrate protein. Thus, in our systems, the formation of co-chaperonin/chaperonin complexes does not necessarily lead to protein folding. By using heterologous oligomer systems, we are able to separate the functions of binding and refolding in order to better understand the chaperonin mechanism.     

Characterisation of mutations in GroES that allow GroEL to function as a single ring

The chaperonin GroEL contains two seven-subunit rings, and allosteric signals between them are required to complete the GroEL reaction cycle. For this reason SR1, a mutant of GroEL that forms only single rings, cannot function as a chaperone. Mutations in SR1 that restore chaperone function weaken its interaction with the cochaperonin GroES. We predicted that GroES mutants with reduced affinity for GroEL would also restore function to SR1. To test this, we mutated residues in GroES in and near its contact site with GroEL. Nearly half of the mutants showed partial function with SR1. Two mutants were confirmed to have reduced affinity for GroEL. Intriguingly, some GroES mutants were able to function with active single ring mutants of GroEL.     

Chaperonin GroESL mediates the protein folding of human liver mitochondrial aldehyde dehydrogenase in Escherichia coli

An efficient bacterial expression system for the human mitochondrial aldehyde dehydrogenase (ALDH2) was developed using co-overexpression of heat shock chaperone gene GroESL. On the basis of the ALDH2 amino acid sequence and cDNA sequences a full-length cDNA encoding wild-type ALDH2 was cloned from a human liver library. A mutant-type ALDH2 (ALDH2(2)) was developed using site-directed mutagenesis of the ALDH2 cDNA and also cloned. Both types of ALDH2 cDNA were subcloned for expression in Escherichia coli (E. coli), recombinant ALDH2 and ALDH2(2) were successfully expressed as soluble active enzymes following co-expression with a second plasmid construct producing GroES and GroEL, E. coli chaperonin proteins. Purified wild-type ALDH2 and mutant ALDH2(2) had a K(m) for acetaldehyde of 0.65 and 25.73 microM, respectively. Co-expression of ALDH2 with ALDH2(2) in the presence of E. coli chaperonins produced a soluble enzyme with a K(m) for acetaldehyde of 8.79 microM, suggesting that the product was a heteromer. Mitochondrial matrix hsp60 and hsp10 chaperonins are then thought to act on imported ALDH2 and are essential for accurate protein folding and multisubunit formation. Protein-protein interactions between ALDH2s and various chaperones were investigated using the yeast two-hybrid system. The wild-type and mutant-type enzymes strongly interacted with each other and GroEL and ALDH2s also interacted but only weakly. Chaperone hsp10 also interacted with hsp60 and ALDH2(1) and ALDH2(2), but again the interactions were weak ones.     

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.     

Combined thermodynamic and kinetic analysis of GroEL interacting with CXCR4 transmembrane peptides

GroEL along with ATP and its co-chaperonin GroES has been demonstrated to significantly enhance the folding of newly translated G-protein-coupled receptors (GPCRs). This work extends the previous studies to explore the guest capture and release processes in GroEL-assisted folding of GPCRs, by the reduced approach of employing CXCR4 transmembrane peptides as model substrates. Each of the CXCR4-derived peptides exhibited high affinity for GroEL with a binding stoichiometry near seven. It is found that the peptides interact with the paired α helices in the apical domain of the chaperonin which are similar with the binding sites of SBP (strongly binding peptide: SWMTTPWGFLHP). Complementary binding study with a single-ring version of GroEL indicates that each of the two chaperonin rings is competent for accommodating all the seven CXCR4 peptides bound to GroEL under saturation condition. Meanwhile, the binding kinetics of CXCR4 peptides with GroEL was also examined; ATP alone, or in combination of GroES evidently promoted the release of the peptide substrates from the chaperonin. The results obtained would be beneficial to understand the thermodynamic and kinetic nature of GroEL-GPCRs interaction which is the central molecular event in the assisted folding process.     

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.     

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.     

The Hsp60-(p.V98I) mutation associated with hereditary spastic paraplegia SPG13 compromises chaperonin function both in vitro and in vivo

We have previously reported the association of a mutation (c.292G > A/p.V98I) in the human HSPD1 gene that encodes the mitochondrial Hsp60 chaperonin with a dominantly inherited form of hereditary spastic paraplegia. Here, we show that the purified Hsp60-(p.V98I) chaperonin displays decreased ATPase activity and exhibits a strongly reduced capacity to promote folding of denatured malate dehydrogenase in vitro. To test its in vivo functions, we engineered a bacterial model system that lacks the endogenous chaperonin genes and harbors two plasmids carrying differentially inducible operons with human Hsp10 and wild-type Hsp60 or Hsp10 and Hsp60-(p.V98I), respectively. Ten hours after shutdown of the wild-type chaperonin operon and induction of the Hsp60-(p.V98I)/Hsp10 mutant operon, bacterial cell growth was strongly inhibited. No globally increased protein aggregation was observed, and microarray analyses showed that a number of genes involved in metabolic pathways, some of which are essential for robust aerobic growth, were strongly up-regulated in Hsp60-(p.V98I)-expressing bacteria, suggesting that the growth arrest was caused by defective folding of some essential proteins. Co-expression of Hsp60-(p.V98I) and wild-type Hsp60 exerted a dominant negative effect only when the chaperonin genes were expressed at relatively low levels. Based on our in vivo and in vitro data, we propose that the major effect of heterozygosity for the Hsp60-(p.V98I) mutation is a moderately decreased activity of chaperonin complexes composed of mixed wild-type and Hsp60-(p.V98I) mutant subunits.