Reactivation of thermally inactivated enzymes by free and immobilized chaperonin GroEL/ES

Thermally inactivated bovine deoxyribonuclease I (DNase I) and yeast enolase were reactivated by GroEL/ES from Escherichia coli. In both cases, GroEL/ES was found to have the ability to reactivate inactivated enzymes in an ATP-dependent manner. GroEL/ES can interact with the enzymes that were denatured at high temperature and convert them to the active conformations. To test the applicability of GroEL/ES to the reactivation processes of thermally inactivated enzymes, GroEL/ES was immobilized using formyl-Cellulofine (GroEL/ES-Cellulofine) and its performance was studied. GroEL/ES-Cellulofine retained a sufficiently high ability to reactivate enzymes. Moreover, GroEL/ES-Cellulofine could be used repeatedly, indicating high durability. These results indicate that immobilized chaperonin is effective for reactivation of enzymes that are thermally inactivated in various bioprocesses. Received: 16 December 1996 / Received last revision: 21 February 1997 / Accepted: 28 February 1997     

Chaperone mediated solubilization of 69-kDa recombinant maltodextrin glucosidase in Escherichia coli

Aims:  To investigate the factors affecting expression and solubilization of Escherichia coli maltodextrin glucosidase in E. coli .
Methods and Results:  Expression level and solubilization of the recombinant E. coli maltodextrin glucosidase was studied in E. coli at different temperatures, in presence of overexpressed GroEL, GroES and externally supplemented glycerol. Aggregation of maltodextrin glucosidase in the cytoplasm was partially prevented by the co-expression of GroEL and GroES, and using externally supplemented glycerol or lowering the culture temperature. Co-expression of GroEL and GroES or simultaneous presence of overexpressed GroEL, GroES and externally supplemented glycerol together resulted significant increase of the activity of maltodextrin glucosidase. The growth rate of E. coli was inhibited by the formation of inclusion bodies whereas the presence of overexpressed GroEL, GroES alone or together with glycerol enhanced the growth rate of E. coli substantially.
Conclusions:  The results indicated that lowering the temperature, use of GroEL, GroES and glycerol could be few controlling factors for the solubilization of recombinant aggregation-prone maltodextrin glucosidase in E. coli.
Significance and Impact of the Study:  Our study could help in developing the strategy for enhancing the production of soluble industrial enzymes and finding the therapeutic agents against protein misfolding diseases.     

Comparative biochemical characterization of two GroEL homologs from the cyanobacterium Synechococcus elongatus PCC 7942

Unlike Escherichia coli, cyanobacteria generally contain two GroEL homologs. The chaperone function of cyanobacterial GroELs was examined in vitro for the first time with GroEL1 and GroEL2 of Synechococcus elongatus PCC 7942. Both GroELs prevented aggregation of heat-denatured proteins. The ATPase activity of GroEL1 was approximately one-sixth that of Escherichia coli GroEL, while that of GroEL2 was insignificant. The activities of both GroELs were enhanced by GroES, while that of Escherichia coli GroEL was suppressed. The ATPase activity of GroEL1 was greatly enhanced in the presence of GroEL2, but the folding activities of GroEL1 and GroEL2 were much lower than that of Escherichia coli GroEL, regardless of the co-presence of the counterpart or GroES. Both native and recombinant GroEL1 forms a tetradecamer like Escherichia coli GroEL, while GroEL2 forms a heptamer or dimer, but the GroEL1 and GroEL2 oligomers were extremely unstable. In sum, we concluded that the cyanobacterial GroELs are mutually distinct and different from Escherichia coli GroEL.     

Effects of the chaperonin GroE on the refolding of tryptophanase from Escherichia coli. Refolding is enhanced in the presence of ADP.

The refolding of the tetrameric enzyme tryptophanase was facilitated by the chaperonin GroE. Maximum refolding yield of tryptophanase molecules (about 80%) was attained in the presence of a 15-fold excess of GroE 21-mer over tryptophanase monomer. The GroEL subunit was required for this improvement in refolding yield, whereas the GroES subunit was not. Light scattering experiments of the refolding reaction revealed that GroE bound to tryptophanase folding intermediates and suppressed their aggregation. The presence of ATP was required for the efficient dissociation of tryptophanase from GroEL. However, our experiments indicated that tryptophanase dissociated readily from GroEL in the presence of not only ATP, but also in the presence of non-hydrolyzable ATP analogues such as ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) and AMP-PNP (adenyl-5'-yl imidodiphosphate) as well. Surprisingly, the release of tryptophanase from GroEL was facilitated in the presence of ADP as well. We concluded that the binding of nucleotides such as ATP and ADP changed the conformation of GroEL and facilitated the dissociation of tryptophanase molecules. The conformation formed in the presence of ADP was distinct from the conformation formed in the presence of ATP, as shown by the selective dissociation of various folding proteins from the two conformations.     

幽门螺杆菌分子伴侣GroEL相互作用蛋白分析

【目的】获得幽门螺杆菌(Helicobacter pylori,HP) GroEL结合蛋白质组构成谱,为进一步探究GroEL及其与相互作用蛋白在HP致病机制中的作用提供新思路。【方法】在构建HP GroEL原核表达重组大肠杆菌(Escherichia coli) BL21(DE3)⁽pET-28a(+)-groEL)基础上,纯化带有His标签的GroEL蛋白,与HP全菌蛋白提取液共孵育后,利用Protein G磁珠和抗His标签抗体免疫沉淀法对复合物进行捕获,然后对复合物中GroEL及其结合的蛋白质进行质谱法鉴定,根据主要功能对其进行分类,并完成蛋白质相互关系网络分析。【结果】对GroEL蛋白捕获成分进行分析,共鉴定出59种可能与GroEL结合的蛋白质,其中包括19种代谢酶类(KatA、GltA和AhpC等参与氧化还原相关酶类7种,PepA、RocF和HtrA等肽酶5种,以及2种参与脂肪代谢酶、2种参与ATP合成酶、2种尿素酶和HP17＿08079蛋白等)、15种外膜蛋白(黏附素BabA、SabA、HapA及其他膜蛋白等)、8种转录翻译相关蛋白(Tuf、RpoBC...     

The interaction of the GroEL chaperone with early kinetic intermediates of renaturing proteins inhibits the formation of their native structure

The main function of the chaperone GroEL is to prevent nonspecific association of nonnative protein chains and provide their correct folding. In the present work, the renaturation kinetics of three globular proteins (human alpha-lactalbumin, bovine carbonic anhydrase, and yeast phosphoglycerate kinase) in the presence of different molar excess of GroEL (up to 10-fold) was studied. It was shown that the formation of the native structure during the refolding of these proteins is retarded with an increase in GroEL molar excess due to the interaction of kinetic protein intermediates with the chaperone. Mg(2+)-ATP and Mg(2+)-ADP weaken this interaction and decrease the retarding effect of GroEL on the protein refolding kinetics. The theoretical modeling of protein folding in the presence of GroEL showed that the experimentally observed linear increase in the protein refolding half-time with increasing molar excess of GroEL must occur only when the protein adopts its native structure outside of GroEL (i.e. in the free state), while the refolding of the protein in the complex with GroEL is inhibited. The dissociation constants of GroEL complexed with the kinetic intermediates of the proteins studied were evaluated, and a simple mechanism of the functioning of GroEL as a molecular chaperone was proposed.     

Denaturation and reassembly of chaperonin GroEL studied by solution X-ray scattering

We measured the denaturation and reassembly of Escherichia coli chaperonin GroEL using small-angle solution X-ray scattering, which is a powerful technique for studying the overall structure and assembly of a protein in solution. The results of the urea-induced unfolding transition show that GroEL partially dissociates in the presence of more than 2 M urea, cooperatively unfolds at around 3 M urea, and is in a monomeric random coil-like unfolded structure at more than 3.2 M urea. Attempted refolding of the unfolded GroEL monomer by a simple dilution procedure is not successful, leading to formation of aggregates. However, the presence of ammonium sulfate and MgADP allows the fully unfolded GroEL to refold into a structure with the same hydrodynamic dimension, within experimental error, as that of the native GroEL. Moreover, the X-ray scattering profiles of the GroEL thus refolded and the native GroEL are coincident with each other, showing that the refolded GroEL has the same structure and the molecular mass as the native GroEL. These results demonstrate that the fully unfolded GroEL monomer can refold and reassemble into the native tetradecameric structure in the presence of ammonium sulfate and MgADP without ATP hydrolysis and preexisting chaperones. Therefore, GroEL can, in principle, fold and assemble into the native structure according to the intrinsic characteristic of its polypeptide chain, although preexisting GroEL would be important when the GroEL folding takes place under in vivo conditions, in order to avoid misfolding and aggregation.     

The interaction of beta(2)-glycoprotein I domain V with chaperonin GroEL: the similarity with the domain V and membrane interaction

To clarify the mechanism of interaction between chaperonin GroEL and substrate proteins, we studied the conformational changes; of the fifth domain of human beta(2)-glycoprotein I upon binding to GroEL. The fifth domain has a large flexible loop, containing several hydrophobic residues surrounded by positively charged residues, which has been proposed to be responsible for the binding of beta(2)-glycoprotein I to negatively charged phospholipid membranes. The reduction by dithiothreitol of the three intramolecular disulfide bonds of the fifth domain was accelerated in the presence of stoichiometric amounts of GroEL, indicating that the fifth domain was destabilized upon interaction with GroEL. To clarify the GroEL-induced destabilization at the atomic level, we performed H/(2)H exchange of amide protons using heteronuclear NMR spectroscopy. The presence of GroEL promoted the H/(2)H exchange of most of the protected amide protons, suggesting that, although the flexible loop of the fifth domain is likely to be responsible for the initiation of binding to GroEL, the interaction with GroEL destabilizes the overall conformation of the fifth domain.     

Effects of divalent cations on encapsulation and release in the GroEL-assisted folding

Chaperonin GroEL assists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL-GroES cis-ternary complexes, but prevent the release of METF from GroEL.     

Synchronized domain-opening motion of GroEL is essential for communication between the two rings

Escherichia coli chaperonin GroEL consists of two stacked rings of seven identical subunits each. Accompanying binding of ATP and GroES to one ring of GroEL, that ring undergoes a large en bloc domain movement, in which the apical domain twists upward and outward. A mutant GroEL(AEX) (C138S,C458S,C519S,D83C,K327C) in the oxidized form is locked in a closed conformation by an interdomain disulfide cross-link and cannot hydrolyze ATP (Murai, N., Makino, Y., and Yoshida, M. (1996) J. Biol. Chem. 271, 28229-28234). By reconstitution of GroEL complex from subunits of both wild-type GroEL and oxidized GroEL(AEX), hybrid GroEL complexes containing various numbers of oxidized GroEL(AEX) subunits were prepared. ATPase activity of the hybrid GroEL containing one or two oxidized GroEL(AEX) subunits per ring was about 70% higher than that of wild-type GroEL. Based on the detailed analysis of the ATPase activity, we concluded that inter-ring negative cooperativity was lost in the hybrid GroEL, indicating that synchronized opening of the subunits in one ring is necessary for the negative cooperativity. Indeed, hybrid GroEL complex reconstituted from subunits of wild-type and GroEL mutant (D398A), which is ATPase-deficient but can undergo domain opening motion, retained the negative cooperativity of ATPase. In contrast, the ability of GroEL to assist protein folding was impaired by the presence of a single oxidized GroEL(AEX) subunit in a ring. Taken together, cooperative conformational transitions in GroEL rings ensure the functional communication between the two rings of GroEL.     

GroEL protects the sarcoplasmic reticulum Ca(++)-dependent ATPase from inactivation in vitro.

The molecular chaperone, GroEL, facilitates correct protein folding and inhibits protein aggregation. The function of GroEL is often, though not invariably, dependent on the co-chaperone, GroES, and ATP. In this study it is shown that GroEL alone substantially reduces the inactivation of purified Ca(++)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum. In the absence of GroEL, the enzyme became completely inactive in about 45-60 hours when kept at 25 degrees C, while in the presence of an equimolar amount of GroEL, the enzyme remained approximately 80% active even after 75 hours. Equimolar amounts of BSA or lysozyme were unable to protect the enzyme from inactivation under identical conditions. Analysis by SDS-PAGE showed GroEL was acting by blocking the aggregation of ATPase at 25 degrees C. GroEL was not as effective in protection at -20 degrees C or 4 degrees C. These results are discussed in the context of current models of the GroEL mechanism.     

Dissociation of the single-ring chaperonin GroEL by high hydrostatic pressure

We investigated the dissociation of single-ring heptameric GroEL (SR1) by high hydrostatic pressure in the range of 1-2.5 kbar. The kinetics of the dissociation of SR1 in the absence and presence of Mg2+, KCl, and nucleotides were monitored using light scattering. The major aim of this investigation was to understand the role of the double-ring structure of GroEL by comparing its dissociation with the dissociation of the single ring. At all the pressures that were studied, SR1 dissociates much faster than the GroEL 14mer. As observed with the GroEL 14mer, SR1 also showed biphasic kinetics and the dissociated monomers do not reassociate readily back to the oligomer. Unlike the GroEL 14mer, the observed rates for SR1 dissociation are independent of the concentrations of Mg2+ and KCl in the studied range. The effects of nucleotides on the observed rates, in the absence or presence of Mg2+ and KCl, are not very significant. The heterogeneity induced in the GroEL molecule with the double-ring structure by ligands such as Mg2+, KCl, and nucleotides is not observed in the case of SR1. This indicates that the inter-ring negative cooperativity in the double-ring GroEL has a major role in this regard. The results presented in this investigation demonstrate that the presence of a second ring in the GroEL 14mer is important for its stability in an environment where the functional ligands of the chaperonin are available.     

The affinity of the GroEL/GroES complex for peptides under conditions of protein folding

The affinity of four short peptides for the Escherichia coli molecular chaperone GroEL was studied in the presence of the co-chaperone GroES and nucleotides. Our data show that binding of GroES to one ring enhances the interaction of the peptides with the opposite GroEL ring, a finding that was related to the structural readjustments in GroEL following GroES binding. We further report that the GroEL/GroES complex has a high affinity for peptides during ATP hydrolysis when protein substrates would undergo repeated cycles of assisted folding. Although we could not determine at which step(s) during the cycle our peptides interacted with GroEL, we propose that successive state changes in GroEL during ATP hydrolysis may create high affinity complexes and ensure maximum efficiency of the chaperone machinery under conditions of protein folding.     

The ATPase activity of GroEL is supported at high temperatures by divalent cations that stabilize its structure

Previously, we reported that the ATPase activity of GroEL that requires potassium and magnesium was highly temperature dependent in the 25–60 °C range. Here, we report that the monovalent cations, rubidium and ammonium were able to fully substitute for potassium; while the divalent cations manganese, cobalt, and nickel supported the ATPase activity of GroEL albeit to a lesser degree than magnesium. ATPase activities with manganese, cobalt, and nickel were 64%, 41%, and 29%, respectively, of the maximum activity (100%) when utilizing magnesium. Interestingly, the ability of all the cations to support the GroEL ATPase activity was somewhat consistent over the entire 25–60 °C range. Maximum ATPase activities were observed at 49 °C. Here, the influence of these cations on the thermal denaturation of GroEL was also monitored using bisANS binding as an indication of the exposure of hydrophobic surfaces during thermal denaturation of GroEL. Maximum exposure of hydrophobic surfaces on GroEL alone or in the presence of each of the monovalent cations was determined to occur at 65 °C. However, the maximum exposure of hydrophobic surfaces on GroEL in the presence of magnesium, manganese, cobalt or nickel was found to occur at 71 °C indicating that GroEL is significantly stabilized against thermal denaturation by these divalent cations.     

Nucleotide-induced transition of GroEL from the high-affinity to the low-affinity state for a target protein: effects of ATP and ADP on the GroEL-affected refolding of alpha-lactalbumin

We studied the refolding kinetics of alpha-lactalbumin in the presence of wild-type GroEL and its ATPase-deficient mutant D398A at various concentrations of nucleotides (ATP and ADP). We evaluated the apparent binding constant between GroEL and the alpha-lactalbumin refolding intermediate quantitatively by numerical simulation analysis of the alpha-lactalbumin refolding curves in the presence and absence of GroEL. The binding constant showed a co-operative decrease with an increase in ATP concentration, whereas the binding constant decreased in a non-co-operative manner with respect to ADP concentration. For the D398A mutant, the ATP-induced decrease in affinity occurred much faster than the steady-state ATP hydrolysis by this mutant, suggesting that ATP binding to GroEL rather than ATP hydrolysis, was responsible for the co-operative decrease in the affinity for the target protein. We thus analyzed the nucleotide-concentration dependence of affinity of GroEL for the target protein using an allosteric Monod-Wyman-Changeux model in which GroEL underwent an ATP-induced co-operative conformational transition between the high-affinity and low-affinity states of the target protein. The transition midpoint of the ATP-induced transition of GroEL has been found to be around 30 microM, in good agreement with the midpoint evaluated in other structural studies of GroEL. The results show that the observed difference between ATP and ADP-induced transitions of GroEL are brought about by a small difference in an allosteric parameter (the ratio of the nucleotide affinities of GroEL in the high-affinity and the low-affinity states), i.e. 4.1 for ATP and 2.6 for ADP.     

Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL.

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition of GroES.     

Multiple structural transitions of the GroEL subunit are sensitive to intermolecular interactions with cochaperonin and refolding polypeptide

In this study we attempted to determine the specific roles of the numerous conformational changes that are observed in the bacterial chaperonin GroEL, by performing stopped-flow experiments on GroEL R231W in the presence of a refolding substrate protein. The apparent rate of one kinetic phase was decreased by approximately 25% in the presence of prebound unfolded malate dehydrogenase while another phase was suppressed completely under the same conditions, reflecting different effects of the unfolded protein on multiple structural transitions within GroEL. The addition of cochaperonin GroES counteracts the effect of the bound substrate protein in the former case, but had no effect on the latter, more extensive suppression. Using a chemically modified form of GroEL R231W which is incapable of releasing substrate proteins at low temperatures, we identified a conformational transition that is implicated in the release of substrate proteins. Parts of the actual process of substrate protein release were also observed through fluorescence resonance energy transfer experiments involving GroEL and labeled substrate protein. Analysis of the energy transfer data revealed an interesting relationship between substrate protein displacement and a specific structural transition in the GroEL apical domain.     

Stability and release requirements of the complexes of GroEL with two homologous mammalian aminotransferases

The mitochondrial (mAAT) and cytosolic (cAAT) homologous isozymes of aspartate aminotransferase are two relatively large proteins that in their nonnative states interact very differently with GroEL. MgATP alone can increase the rate of GroEL-assisted reactivation of cAAT, yet the presence of GroES is mandatory for mAAT. Addition of an excess of a denatured substrate accelerates reactivation of cAAT in the presence of GroEL, but has no effect on mAAT. These competition studies suggest that the more stringent substrate mAAT forms a thermodynamically stable complex with GroEL, while rebinding affects the slow reactivation kinetics of cAAT with GroEL alone. However, the competitor appears to accelerate the release of cAAT from GroEL, most likely by displacing bound cAAT from the GroEL cavity. Moreover, cAAT, but not mAAT, shows a time-dependent increase in protease resistance while bound to GroEL at low temperature. These results suggest that folding and release of cAAT from GroEL in the absence of cofactors may occur stepwise with certain interactions being broken and reformed until the protein escapes binding. The distinct behavior of these two isozymes most likely results from differences in the structure of the nonnative states that bind to GroEL.     

Activation and expression of proteins during synchronous germination of aerial spores of Streptomyces granaticolor

Synchronously germinating aerial spores of Streptomyces granaticolor were used to study protein activation and expression during the transition from dormant to metabolically active vegetative forms. The first phase of protein activation is associated with the solubility of proteins. Three major chaperones, DnaK, Trigger factor, and GroEL, were identified in spores. Enhancement in rate of protein synthesis during germination was accompanied by the association of TF and DnaK with ribosomes. During germination, the chaperones TF, GroEL, and DnaK undergo reversible phosphorylation. GroEL was phosphorylated on both Ser and Thr, whereas phosphorylation of DnaK and TF was detected on Thr only. A proteomic approach was used to gain more information on protein expression during germination on two types of media differing in the ability of cells to produce antibiotic granaticin. To obtain an overview of the metabolic activity of germinating spores, glycolytic enzymes, enzymes of citric acid cycle, metabolism of amino acids and nucleic acids, and components of the protein synthesis system were identified and analyzed using the proteomic database. The results were deposited on the SWICZ proteomic server and are accessible on http://proteom.biomed.cas.cz.     

Activity of chloramphenicol acetyltransferase overproduced in E. coli with wild-type and mutant GroEL.

The homo-oligomeric protein chloramphenicol acetyltransferase (CAT) has previously been shown to interact with a chaperone GroEL in vitro, suggesting a possible involvement of GroEL in CAT assembly. CAT was overproduced to various levels in the presence and absence of GroEL overproduction, and in groEL mutants. CAT was accumulated to 9-45% of total cellular protein in a fully soluble form, without formation of inclusion bodies. In all cases, even with groEL mutants, CAT specific activity was shown proportional to the amount of protein produced, indicating the formation of active trimer CAT structure does not need GroEL in Escherichia coli.