GroEL–GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli

Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL–ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL–GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL–ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL–ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli.     

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.     

GroEL/GroES-mediated folding of a protein too large to be encapsulated.

The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered. We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release. Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES.     

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

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

Essential role of the chaperonin folding compartment in vivo

The GroEL/GroES chaperonin system of Escherichia coli forms a nano-cage allowing single protein molecules to fold in isolation. However, as the chaperonin can also mediate folding independently of substrate encapsulation, it remained unclear whether the folding cage is essential in vivo. To address this question, we replaced wild-type GroEL with mutants of GroEL having either a reduced cage volume or altered charge properties of the cage wall. A stepwise reduction in cage size resulted in a gradual loss of cell viability, although the mutants bound non-native protein efficiently. Strikingly, a mild reduction in cage size increased the yield and the apparent rate of green fluorescent protein folding, consistent with the view that an effect of steric confinement can accelerate folding. As shown in vitro, the observed acceleration of folding was dependent on protein encapsulation by GroES but independent of GroES cycling regulated by the GroEL ATPase. Altering the net-negative charge of the GroEL cage wall also strongly affected chaperonin function. Based on these findings, the GroEL/GroES compartment is essential for protein folding in vivo.     

Low temperature of GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of deltatigdeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C deltatigdeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of deltatigdeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/= 60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

A comparison of the GroE chaperonin requirements for sequentially and structurally homologous malate dehydrogenases: the importance of folding kinetics and solution environment

Escherichia coli malate dehydrogenase (EcMDH) and its eukaryotic counterpart, porcine mitochondrial malate dehydrogenase (PmMDH), are highly homologous proteins with significant sequence identity (60%) and virtually identical native structural folds. Despite this homology, EcMDH folds rapidly and efficiently in vitro and does not seem to interact with GroE chaperonins at physiological temperatures (37 degrees C), whereas PmMDH folds much slower than EcMDH and requires these chaperonins to fold to the native state at 37 degrees C. Double jump experiments indicate that the slow folding behavior of PmMDH is not limited by proline isomerization. Although the folding enhancer glycerol (<5 m) does not alter the renaturation kinetics of EcMDH, it dramatically accelerates the spontaneous renaturation of PmMDH at all temperatures tested. Kinetic analysis of PmMDH renaturation with increasing glycerol concentrations suggests that this osmolyte increases the on-pathway kinetics of the monomer folding to assembly-competent forms. Other osmolytes such as trimethylamine N-oxide, sucrose, and betaine also reactivate PmMDH at nonpermissive temperatures (37 degrees C). Glycerol jump experiments with preformed GroEL.PmMDH complexes indicate that the shift between stringent (requires ATP and GroES) and relaxed (only requires ATP) complex conformations is rapid (<3-5 s). The similarity in irreversible misfolding kinetics of PmMDH measured with glycerol or the activated chaperonin complex (GroEL.GroES.ATP) suggests that these folding aids may influence the same step in the PmMDH folding reaction. Moreover, the interactions between glycerol-induced PmMDH folding intermediates and GroEL.GroES.ATP are diminished. Our results support the notion that the protein folding kinetics of sequentially and structurally homologous proteins, rather than the structural fold, dictates the GroE chaperonin requirement.     

Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of DeltatigDeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C DeltatigDeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of DeltatigDeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/=60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Purification and characterization of the GroESLx chaperonins from the symbiotic X-bacteria in Amoeba proteus.

GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized sigma(70)-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37 degrees C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37 degrees C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37 degrees C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx(7) and GroELx(14) in two stacks of GroELx(7). In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.     

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

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

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

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

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.     

Filamentous morphology in GroE-depleted Escherichia coli induced by impaired folding of FtsE

The chaperonin GroE (GroEL and the cochaperonin GroES) is the only chaperone system that is essential for the viability of Escherichia coli. It is known that GroE-depleted cells exhibit a filamentous morphology, suggesting that GroE is required for the folding of proteins involved in cell division. Although previous studies, including proteome-wide analyses of GroE substrates, have suggested several targets of GroE in cell division, there is no direct in vivo evidence to identify which substrates exhibit obligate dependence on GroE for folding. Among the candidate substrates, we found that prior excess production of FtsE, a protein engaged in cell division, completely suppressed the filamentation of GroE-depleted E. coli. The GroE depletion led to a drastic decrease in FtsE, and the cells exhibited a known phenotype associated with impaired FtsE function. In the GroE-depleted filamentous cells, the localizations of FtsA and ZipA, both of which assemble with the FtsZ septal ring before FtsE, were normal, whereas FtsX, the interaction partner of FtsE, and FtsQ, which is recruited after FtsE, did not localize to the ring, suggesting that the decrease in FtsE is a cause of the filamentous morphology. Finally, a reconstituted cell-free translation system revealed that the folding of newly translated FtsE was stringently dependent on GroEL/GroES. Based on these findings, we concluded that FtsE is a target substrate of the GroE system in E. coli cell division.     

Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and bacteriophage T4's Gp31

Bacteriophage T4-encoded Gp31 is a functional ortholog of the Escherichia coli GroES cochaperonin protein. Both of these proteins form transient, productive complexes with the GroEL chaperonin, required for protein folding and other related functions in the cell. However, Gp31 is specifically required, in conjunction with GroEL, for the correct folding of Gp23, the major capsid protein of T4. To better understand the interaction between GroEL and its cochaperonin cognates, we determined whether the so-called "pseudo-T-even bacteriophages" are dependent on host GroEL function and whether they also encode their own cochaperonin. Here, we report the isolation of an allele-specific mutation of bacteriophage RB49, called epsilon22, which permits growth on the E. coli groEL44 mutant but not on the isogenic wild type host. RB49 epsilon22 was used in marker rescue experiments to identify the corresponding wild type gene, which we have named cocO (cochaperonin cognate). CocO has extremely limited identity to GroES but is 34% identical and 55% similar at the protein sequence level to T4 Gp31, sharing all of the structural features of Gp31 that distinguish it from GroES. CocO can substitute for Gp31 in T4 growth and also suppresses the temperature-sensitive phenotype of the E. coli groES42 mutant. CocO's predicted mobile loop is one residue longer than that of Gp31, with the epsilon22 mutation resulting in a Q36R substitution in this extra residue. Both the CocO wild type and epsilon22 proteins have been purified and shown in vitro to assist GroEL in the refolding of denatured citrate synthase.     

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.     

Proteomic analysis of heat-stable proteins in Escherichia coli

Some proteins of E. coli are stable at temperatures significantly higher than 49 degrees C, the maximum temperature at which the organism can grow. The heat stability of such proteins would be a property which is inherent to their structures, or it might be acquired by evolution for their specialized functions. In this study, we describe the identification of 17 heat-stable proteins from E. coli. Approximately one-third of these proteins were recognized as having functions in the protection of other proteins against denaturation. These included chaperonin (GroEL and GroES), molecular chaperones (DnaK and FkpA) and peptidyl prolyl isomerases (trigger factor and FkpA). Another common feature was that five of these proteins (GroEL, GroES, Ahpc, RibH and ferritin) have been shown to form a macromolecular structure. These results indicated that the heat stability of certain proteins may have evolved for their specialized functions, allowing them to cope with harsh environments, including high temperatures.     

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.     

Chaperonin GroEL meets the substrate protein as a "load" of the rings

Chaperonin GroEL is an essential molecular chaperone that assists protein folding in the cell. With the aid of cochaperonin GroES and ATP, double ring-shaped GroEL encapsulates non-native substrate proteins inside the cavity of the GroEL-ES complex. Although extensive studies have revealed the outline of GroEL mechanism over the past decade, central questions remain: What are the in vivo substrate proteins? How does GroEL encapsulate the substrates inside the cavity in spite of an apparent entropic difficulty? Is the folding inside the GroEL-ES cavity the same as bulk spontaneous folding? In this review I summarize the recent progress on in vivo and in vitro aspects of GroEL. In particular, emerging evidence shows that the substrate protein itself influences the chaperonin GroEL structure and reaction cycle. Finally I propose the mechanistic similarity between GroEL and kinesin, a molecular motor that moves along a microtubule in an ATP-dependent manner.