Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.3.1 (citrate synthase)
4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.
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PMID:GroE facilitates refolding of citrate synthase by suppressing aggregation. 167 55

The mechanism by which correctly folded proteins are recovered from stable complexes with groEL is not well understood. Certain target proteins require ATP and groES, while others seemingly dispense with the cochaperonin. Here, we examine the chaperonin-assisted folding of ribulose-1,5-bisphosphate carboxylase, malate dehydrogenase, and citrate synthase, three proteins that are believed to require both chaperonin components for successful reactivation. Surprisingly, in all cases, the need for groES depended on the folding environment. Under "non-permissive" conditions, where unassisted spontaneous folding could not occur, reactivation to the native state required the complete chaperonin system (e.g. groEL, groES, and MgATP). However, under "permissive" conditions where spontaneous folding could occur groES was no longer mandatory. Instead, upon the addition of ATP alone, all three target proteins could be released from groEL, in a form that was capable of reaching the native state. In the permissive setting, groES merely accelerated the rate of the ATP-dependent release process. The results suggest that the incompletely folded protein species that are released from groEL, in the absence of groES, are not necessarily committed to the native state. Similar to the unassisted folding reaction, they still partition between productive and unproductive folding pathways in an environment-dependent manner. It follows that the mechanistic contribution of the co-chaperonin, groES, and its physiological significance in cellular protein folding, could be entirely missed in a permissive in vitro environment.
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PMID:On the role of groES in the chaperonin-assisted folding reaction. Three case studies. 790 92

The prokaryotic molecular chaperone GroE is increasingly expressed under heat shock conditions. GroE protects cells by preventing the irreversible aggregation of thermally unfolding proteins. Here, the interaction of GroE with thermally unfolding citrate synthase (CS) was dissected into several steps that occur before irreversible aggregation, and the conformational states of the unfolding protein recognized by GroEL were determined. The kinetic analysis of CS unfolding revealed the formation of inactive dimeric and monomeric intermediates. GroEL binds both intermediates without affecting the unfolding pathway. Furthermore, the dimeric intermediates are not protected against dissociation in the presence of GroEL. Monomeric CS is stably associated with GroEL, thus preventing further irreversible unfolding steps and subsequent aggregation. During refolding, monomeric CS is encapsulated inside the cavity of GroEL. GroES complexes. Taken together our results suggest that for protection of cells against heat stress both the ability of GroEL to interact with a large variety of nonnative conformations of proteins and the active, GroES-dependent refolding of highly unfolded species are important.
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PMID:GroEL traps dimeric and monomeric unfolding intermediates of citrate synthase. 983 3

Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.
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PMID:The production and characterisation of an immobilised chaperonin system. 987 99

Chloroplast chaperonin 20 (Cpn20) in higher plants is a functional homologue of the Escherichia coli GroES, which is a critical regulator of chaperonin-mediated protein folding. The cDNA for a Cpn20 homologue of Arabidopsis thaliana was isolated. It was 958 bp long, encoding a protein of 253 amino acids. The protein was composed of an N-terminal chloroplast transit peptide, and the predicted mature region comprised two distinct GroES domains that showed 42% amino acid identity to each other. The isolated cDNA was constitutively expressed in transgenic tobacco. Immunogold labelling showed that Cpn20 is accumulated in chloroplasts of transgenic tobacco. A Northern blot analysis revealed that mRNA for the chloroplast Cpn20 is abundant in leaves and is increased by heat treatment. To examine the oligomeric structure of Cpn20, a histidine-tagged construct lacking the transit peptide was expressed in E. coli and purified by affinity chromatography. Gel-filtration and cross-linking analyses showed that the expressed products formed a tetramer. The expressed products could substitute for GroES to assist the refolding of citrate synthase under non-permissive conditions. The analysis on the subunit stoichiometry of the GroEL-Cpn20 complex also revealed that the functional complex is composed of a GroEL tetradecamer and a Cpn20 tetramer.
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PMID:Chloroplast Cpn20 forms a tetrameric structure in Arabidopsis thaliana. 1020 3

The molecular chaperones GroEL and GroES facilitate protein folding in an ATP-dependent manner under conditions where no spontaneous folding occurs. It has remained unknown whether GroE achieves this by a passive sequestration of protein inside the GroE cavity or by changing the folding pathway of a protein. Here we used citrate synthase, a well studied model substrate, to discriminate between these possibilities. We demonstrate that GroE maintains unfolding intermediates in a state that allows productive folding under nonpermissive conditions. During encapsulation of non-native protein inside GroEL.GroES complexes, a folding reaction takes place, generating association-competent monomeric intermediates that are no longer recognized by GroEL. Thus, GroE shifts folding intermediates to a productive folding pathway under heat shock conditions where even the native protein unfolds in the absence of GroE.
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PMID:Analysis of GroE-assisted folding under nonpermissive conditions. 1040 Jun 32

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.
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PMID:Pseudo-T-even bacteriophage RB49 encodes CocO, a cochaperonin for GroEL, which can substitute for Escherichia coli's GroES and bacteriophage T4's Gp31. 1110 67

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 mug/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.
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PMID:Efficient refolding of aggregation-prone citrate synthase by polyol osmolytes: how well are protein folding and stability aspects coupled? 1569 14

Molecular chaperones play a central role in maintaining protein structure within a cell. Previously, we determined that the gene encoding a molecular chaperone, a thermosome, from the hyperthermophilic archaeon Methanocaldococcus jannaschii is upregulated upon lethal heat shock. We have recombinantly expressed this thermosome (rTHS) and show here that it is both stable and fully functional in aqueous solutions containing water-miscible organic co-solvents. Based on circular dichroism the secondary structure of rTHS was not affected by one-hour exposures to a variety of co-solvents including 30% v/v acetonitrile (ACN) and 50% methanol (MeOH). By contrast, the secondary structure of a mesophilic homologue, GroEL/GroES (GroE), was substantially disrupted. rTHS reduced the aggregation of ovalbumin and citrate synthase in 30% ACN, assisted refolding of citrate synthase upon solvent-inactivation, and stabilized citrate synthase and glutamate dehydrogenase in the direct presence of co-solvents. Apparent total turnover numbers of these enzymes in denaturing solutions increased by up to 2.5-fold in the presence of rTHS. Mechanistic models are proposed to help ascertain specific conditions that could enhance or limit organic solvent-induced chaperone activity. These models suggest that thermodynamic stability and the reversibility of enzyme unfolding play key roles in the effectiveness of enzyme recovery by rTHS.
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PMID:Chaperone function in organic co-solvents: experimental characterization and modeling of a hyperthermophilic chaperone subunit from Methanocaldococcus jannaschii. 1815 40

The bacterial chaperonin GroEL and the co-chaperonin GroES assist in the folding of a number of structurally unrelated substrate proteins (SPs). In the absence of chaperonins, SP folds by the kinetic partitioning mechanism (KPM), according to which a fraction of unfolded molecules reaches the native state directly, while the remaining fraction gets trapped in a potentially aggregation-prone misfolded state. During the catalytic reaction cycle, GroEL undergoes a series of allosteric transitions (T<-->R-->R"-->T) triggered by SP capture, ATP binding and hydrolysis, and GroES binding. We developed a general kinetic model that takes into account the coupling between the rates of the allosteric transitions and the folding and aggregation of the SP. Our model, in which the GroEL allosteric rates and SP-dependent folding and aggregation rates are independently varied without prior assumption, quantitatively fits the GroEL concentration-dependent data on the yield of native ribulose bisphosphate carboxylase/oxygenase (Rubisco) as a function of time. The extracted kinetic parameters for the GroEL reaction cycle are consistent with the available values from independent experiments. In addition, we also obtained physically reasonable parameters for the kinetic steps in the reaction cycle that are difficult to measure. If experimental values for GroEL allosteric rates are used, the time-dependent changes in native-state yield at eight GroEL concentrations can be quantitatively fit using only three SP-dependent parameters. The model predicts that the differences in the efficiencies (as measured by yields of the native state) of GroEL, single-ring mutant (SR1), and variants of SR1, in the rescue of mitochondrial malate dehydrogenase, citrate synthase, and Rubisco, are related to the large variations in the allosteric transition rates. We also show that GroEL/S mutants that efficiently fold one SP at the expense of all others are due to a decrease in the rate of a key step in the reaction cycle, which implies that wild-type GroEL has evolved as a compromise between generality and specificity. We predict that, under maximum loading conditions and saturating ATP concentration, the efficiency of GroEL (using parameters for Rubisco) depends predominantly on the rate of R-->R" transition, while the equilibrium constant of the T<-->R has a small effect only. Both under sub- and superstoichiometric GroEL concentrations, enhanced efficiency is achieved by rapid turnover of the reaction cycle, which is in accord with the predictions of the iterative annealing mechanism. The effects are most dramatic at substoichiometric conditions (most relevant for in vivo situations) when SP aggregation can outcompete capture of SP by chaperonins.
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PMID:Kinetic model for the coupling between allosteric transitions in GroEL and substrate protein folding and aggregation. 1831 71


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