Preformed GroES Oligomers Are Not Required as Functional Cochaperonins

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268–30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96–97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer–monomer equilibrium to be 7.3×10^–34M⁶. The GroESD(96–97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96–97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the preformed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.     

Single-molecule Study on the Decay Process of the Football-shaped GroEL-GroES Complex Using Zero-mode Waveguides

It has been widely believed that an asymmetric GroEL-GroES complex (termed the bullet-shaped complex) is formed solely throughout the chaperonin reaction cycle, whereas we have recently revealed that a symmetric GroEL-(GroES)₂ complex (the football-shaped complex) can form in the presence of denatured proteins. However, the dynamics of the GroEL-GroES interaction, including the football-shaped complex, is unclear. We investigated the decay process of the football-shaped complex at a single-molecule level. Because submicromolar concentrations of fluorescent GroES are required in solution to form saturated amounts of the football-shaped complex, single-molecule fluorescence imaging was carried out using zero-mode waveguides. The single-molecule study revealed two insights into the GroEL-GroES reaction. First, the first GroES to interact with GroEL does not always dissociate from the football-shaped complex prior to the dissociation of a second GroES. Second, there are two cycles, the “football cycle ” and the “bullet cycle,” in the chaperonin reaction, and the lifetimes of the football-shaped and the bullet-shaped complexes were determined to be 3–5 s and about 6 s, respectively. These findings shed new light on the molecular mechanism of protein folding mediated by the GroEL-GroES chaperonin system.     

GroEL assisted folding of large polypeptide substrates in Escherichia coli: Present scenario and assignments for the future

Escherichia coli chaperonins GroEL and GroES are indispensable for survival and growth of the cell since they provide essential assistance to the folding of many newly translated proteins in the cell. Recent studies indicate that a substantial portion of the proteins involved in the host pathways are completely dependent on GroEL–GroES for their folding and hence providing some explanation for why GroEL is essential for cell growth. Many proteins either small-single domain or large multidomains require assistance from GroEL–ES during their lifetime. Proteins of size up to 70 kDa can fold via the cis mechanism during GroEL–ES assisted pathway, but other proteins (>70 kDa) that cannot be pushed inside the cavity of GroEL–ATP complex upon binding of GroES fold by an evolved mechanism called trans. In recent years, much work has been done on revealing facts about the cis mechanism involving the GroEL assisted folding of small proteins whereas the trans mechanism with larger polypeptide substrates still remains under cover. In order to disentangle the role of chaperonin GroEL–GroES in the folding of large E. coli proteins, this review discusses a number of issues like the range of large polypeptide substrates acted on by GroEL. Do all these substrates need the complete chaperonin system along with ATP for their folding? Does GroEL act as foldase or holdase during the process? We conclude with a discussion of the various queries that need to be resolved in the future for an extensive understanding of the mechanism of GroEL mediated folding of large substrate proteins in E. coli cytosol.     

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 σ⁷⁰-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37°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°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°C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx₇ and GroELx₁₄ in two stacks of GroELx₇. 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.     

Asp-52 in Combination with Asp-398 Plays a Critical Role in ATP Hydrolysis of Chaperonin GroEL

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼150 h (∼6 days), providing a good model to characterize the football-shaped complex.     

Transmission Electron Microscopy of GroEL, GroES, and the Symmetrical GroEL/ES Complex

Two new 2-D crystal forms of the Escherichia coli chaperone GroEL (cpn60) 2 × 7-mer have been produced using the negative staining-carbon film (NS-CF) technique. These 2-D crystals, which contain the cylindrical GroEL in side-on and end-on orientations, both possess p21 symmetry, with two molecules in the respective unit cells. The crystallographically averaged images correlate well with those obtained by other authors from single particle analysis of GroEL and our own previous crystallographic analysis. 2-D crystallization of the smaller chaperone GroES (cpn10) 7-mer has also been achieved using the NS-CF technique. Crystallographically averaged images of GroES single particle images indicate considerable variation in molecular shape, which is most likely due to varying molecular orientation on the carbon support film. The quaternary structure of GroES does, nevertheless, approximate to a ring-like shape. The complex formed by GroEL and GroES in the presence of ATP at room temperature has been shown to possess a symmetrical hollow ellipsoidal conformation. This symmetrical complex forms in the presence of a 2:1 or greater molar ratio of GroES:GroEL. At lower molar ratios linear chains of GroEL form, apparently linked by GroES in a 1:1 manner, which provide supportive evidence for the ability of both ends of the GroEL cylinder to interact with GroES. The apparent discrepancy between our data and that of other groups who have described an asymmetrical "bullet-shaped" (holo-chaperone) GroEL/ES complex is discussed in detail.     

Molecular characterization of GroES and GroEL homologues from Clostridium botulinum

We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947). The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies. Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue. The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced. The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins. Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage.     

Evolution of Escherichia coli for Growth at High Temperatures

Evolution depends on the acquisition of genomic mutations that increase cellular fitness. Here, we evolved Escherichia coli MG1655 cells to grow at extreme temperatures. We obtained a maximum growth temperature of 48.5 °C, which was not increased further upon continuous cultivation at this temperature for >600 generations. Despite a permanently induced heat shock response in thermoresistant cells, only exquisitely high GroEL/GroES levels are essential for growth at 48.5 °C. They depend on the presence of lysyl-tRNA-synthetase, LysU, because deletion of lysU rendered thermoresistant cells thermosensitive. Our data suggest that GroEL/GroES are especially required for the folding of mutated proteins generated during evolution. GroEL/GroES therefore appear as mediators of evolution of extremely heat-resistant E. coli cells.     

分子伴侣GroESL在大肠杆菌中的克隆、表达及分离纯化

将含分子伴侣GroESL基因的DNA片段,通过在合适的酶切位点进行酶切,将该基因克隆入高表达载体pKC220,含该表达质粒的工程菌经42℃热诱导后,分子伴侣GroESL基因在大肠杆菌中获得了高效表达,其中,分子伴侣蛋白GroEL的表达量占细菌总蛋白的40％,其辅助蛋白GroES的表达量占细菌总蛋白的15％;同时,建立了较简单的分离纯化路线,通过硫酸铵沉淀、DEAE-52柱层析,Sephadex G-50凝胶过滤等方法得到纯化的分子伴侣蛋白GroEL和GroES。     

Expression of a Highly Toxic Protein, Bax, in Escherichia coli by Attachment of a Leader Peptide Derived from the GroES Cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384–11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

Bacteriophage-encoded cochaperonins can substitute for Escherichia coli's essential GroES protein

The Escherichia coli chaperonin machine is composed of two members, GroEL and GroES. The GroEL chaperonin can bind 10–15% of E. coli’s unfolded proteins in one of its central cavities and help them fold in cooperation with the GroES cochaperonin. Both proteins are absolutely essential for bacterial growth. Several large, lytic bacteriophages, such as T4 and RB49, use the host-encoded GroEL in conjunction with their own bacteriophage-encoded cochaperonin for the correct assembly of their major capsid protein, suggesting a cochaperonin specificity for the in vivo folding of certain substrates. Here, we demonstrate that, when the cochaperonin of either bacteriophage T4 (Gp31) or RB49 (CocO) is expressed in E. coli, the otherwise essential groES gene can be deleted. Thus, it appears that, despite very little sequence identity with groES, the bacteriophage-encoded Gp31 and CocO proteins are capable of replacing GroES in the folding of E. coli’s essential, housekeeping proteins.     

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.     

Probing Open Conformation of GroEL Rings by Cross-linking Reveals Single and Double Open Ring Structures of GroEL in ADP and ATP

Two heptamer rings of chaperonin GroEL undergo opening-closing conformational transition in the reaction cycle with the aid of GroES and ATP. We introduced Cys into the GroEL subunit at Ala-384 and Ser-509, which are very close between adjacent GroEL subunits in the open heptamer ring but far apart in the closed heptamer ring. The open ring-specific inter-subunit cross-linking between these Cys indicated that the number of rings in open conformation in GroEL was two in ATP (GroEL_OO), one in ADP (GroEL_O), and none in the absence of nucleotide. ADP showed an inhibitory effect on ATP-induced generation of GroEL_OO. The isolated GroEL_O and GroEL_OO, which lost any bound nucleotide, could bind GroES to form a bullet-shaped 1:1 GroEL-GroES complex and a football-shaped 1:2 GroEL-GroES complex, respectively, even without the addition of any nucleotide. Substrate protein was unable to form a stable complex with GroEL_OO and did not stimulate ATPase activity of GroEL. These results favor a model of the GroEL reaction cycle that includes a football complex as a critical intermediate.Chaperonin facilitates the folding of other proteins using the energy of ATP hydrolysis (1–4). GroEL, an Escherichia coli chaperonin, consists of 14 identical 57-kDa subunits arranged in two heptamer rings. Each ring contains a central open cavity, and the two rings are stacked back-to-back (5). Denatured protein binds to the apical end of the central cavity of the heptamer ring of GroEL (6–10). In the presence of ATP, a disk-shaped GroES binds to the same apical end as a lid to seal the cavity and generates a chamber. The denatured protein is discharged into the chamber, making this heptamer ring folding-active, where productive folding proceeds (11, 12). After several seconds, the GroES lid is detached from GroEL, and the substrate protein is free to escape into solution.Two heptamer rings of GroEL undergo opening-closing conformational transition, coupled with attachment and detachment of GroES, in the functional cycle (13). In the transition from “closed” to “open” conformation, apical domain of each GroEL subunit in the ring is shifted upward and outward, and the cleft between apical and equatorial domains opens. GroES is associated with the open ring, and two kinds of GroEL-GroES complexes are formed. An asymmetric “bullet”-shaped complex is a 1:1 GroEL-GroES complex in which GroES attached to one of two heptamer rings in GroEL (14–16). A symmetric “football”-shaped complex is a 1:2 GroEL-GroES complex in which GroES attached to both heptamer rings of GroEL (17–22). The football complex contains two open rings; the bullet complex contains one closed and one open ring, and free GroEL is made up of two closed rings.Previously, we generated the GroEL in which two rings in GroEL were locked in a closed conformation by disulfide cross-link between apical and equatorial domains in the same GroEL subunits (23). This GroEL can bind ATP and denatured protein but fails to process further reaction steps such as ATP hydrolysis, GroES binding, and release of substrate protein. We report here the opposite version; open conformation-specific inter-subunit cross-links were introduced into the GroEL ring. Using this cross-linking as a probe of open conformation, we found that one ring was open in ADP (GroEL_O), although two rings were open in ATP (GroEL_OO). The isolated GroEL_O and GroEL_OO, which were nucleotide-free, formed a stable bullet and football complex with GroES even in the absence of any nucleotide. These results support a GroEL mechanism that includes a football complex as a critical intermediate.     

Cloning and expression of soluble recombinant protein comprising the extracellular domain of the human type I interferon receptor 2c subunit (IFNAR-2c) in E. coli

An optimized procedure was developed for production of the extracellular domain encoding amino acids 1–243 of the human type I interferon receptor 2c subunit (IFNAR-2c) as a fusion protein with glutathione S-transferase (GST-IFNAR2cEC) in E. coli to obtain active, soluble protein. Induction of protein expression at 37 °C resulted in a formation of inclusion body. Co-expression with bacterial chaperones, GroEL and GroES, failed to support the folding of GST-IFNAR2cEc under IPTG induction at 37 °C. Expression induced at 25 °C decreased the inclusion body formation up to 62% and cell disruption by a French press provided higher recovery of the recombinant protein than cell disruption by sonication.     

In vivo suppression of phytochrome aggregation by the GroE chaperonins in Escherichia coli

When expressed in Escherichia coli, a truncated form of phytochrome (oat PHYA AP3 residues 464-1129) self associates to form a series of products ranging in size from monomers to aggregates of greater than 20 subunits. When these same phytochrome sequences are coexpressed with the chaperonins GroEL and GroES, the truncated phytochrome migrates as a native-like dimer in size exclusion chromatography and no higher-order aggregates were detected. GroEL and GroES inhibition of phytochrome aggregation in E. coli presumably occurs via the suppression of folding pathways leading to incorrectly folded phytochrome.     

Molecular chaperone GroEL/ES: Unfolding and refolding processes

Molecular chaperones are a special class of heat shock proteins (Hsp) that assist the folding and formation of the quaternary structure of other proteins both in vivo and in vitro. However, some chaperones are complex oligomeric proteins, and one of the intriguing questions is how the chaperones fold. The representatives of the Escherichia coli chaperone system GroEL (Hsp60) and GroES (Hsp10) have been studied most intensively. GroEL consists of 14 identical subunits combined into two interacting ring-like structures of seven subunits each, while the co-chaperone GroES interacting with GroEL consists of seven identical subunits combined into a dome-like oligomeric structure. In spite of their complex quaternary structure, GroEL and GroES fold well both in vivo and in vitro. However, the specific oligomerization of GroEL subunits is dependent on ligands and external conditions. This review analyzes the literature and our own data on the study of unfolding (denaturation) and refolding (renaturation) processes of these molecular chaperones and the effect of ligands and solvent composition. Such analysis seems to be useful for understanding the folding mechanism not only of the GroEL/GroES complex, but also of other oligomeric protein complexes.     

Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8 Å Reveals Rearrangement between Two GroEL Rings

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES₂ complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES₂-ATP₁₄ complex from Escherichia coli at 3.8 Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~ 7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.     

Elucidation of steps in the capture of a protein substrate for efficient encapsulation by GroE

We have identified five structural rearrangements in GroEL induced by the ordered binding of ATP and GroES. The first discernable rearrangement (designated T --> R(1)) is a rapid, cooperative transition that appears not to be functionally communicated to the apical domain. In the second (R(1) --> R(2)) step, a state is formed that binds GroES weakly in a rapid, diffusion-limited process. However, a second optical signal, carried by a protein substrate bound to GroEL, responds neither to formation of the R(2) state nor to the binding of GroES. This result strongly implies that the substrate protein remains bound to the inner walls of the initially formed GroEL.GroES cavity, and is not yet displaced from its sites of interaction with GroEL. In the next rearrangement (R(2).GroES --> R(3).GroES) the strength of interaction between GroEL and GroES is greatly enhanced, and there is a large and coincident loss of fluorescence-signal intensity in the labeled protein substrate, indicating that there is either a displacement from its binding sites on GroEL or at least a significant change of environment. These results are consistent with a mechanism in which the shift in orientation of GroEL apical domains between that seen in the apo-protein and stable GroEL.GroES complexes is highly ordered, and transient conformational intermediates permit the association of GroES before the displacement of bound polypeptide. This ensures efficient encapsulation of the polypeptide within the GroEL central cavity underneath GroES.     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.