Hydrolysable ATP is a requirement for the correct interaction of molecular chaperonins cpn60 and cpn10.
ABSTRACT: Over recent years the binding ability of the molecular chaperone cpn60 (GroEL14) and its co-chaperone cpn10 (GroES7) has been reported to occur under an assortment of specific conditions from the use of non-hydrolysable ATP analogues (namely adenosine 5'-[gamma-thio]triphosphate) to requiring hydrolysable ATP for any interaction to occur. We have investigated this further using the molecular hydrodynamic methods (hydrodynamic bead modelling, sedimentation-velocity analytical ultracentrifugation and dynamic light-scattering), allowing the process to be followed under physiologically relevant dilute solution conditions, combined with absorption spectrophotometry to determine GroES7-GroEL14 interaction through the rate inhibition of the cpn60's ATPase activity by GroES7. The results found here indicate that the presence of hydrolysable ATP is required to facilitate correct GroES7 interaction with GroEL14 in solution.
Project description:In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence and absence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). The refolded yields of active malate dehydrogenase are increased almost 3-fold in the presence of groEL, groES, Mg2+/ATP and K+ ions. Chaperonin-assisted refolding of malate dehydrogenase does not have an absolute requirement for K+ ions but Mg2+/ATP is obligatory. When ATP is replaced by other nucleoside triphosphates, or by non-hydrolysable ATP analogues, assisted refolding is prevented. Optimal chaperonin-assisted refolding requires both groEL and groES homo-oligomers in molar excess over malate dehydrogenase. Kinetic analysis shows that the chaperonins do not catalyse the refolding of malate dehydrogenase but increase the flux of unfolded enzyme through the productive refolding pathway without altering and/or accelerating that pathway. Although not acting as refolding catalysts, the chaperonins are able to assist at least six consecutive cycles of malate dehydrogenase refolding.
Project description:The groE operon of Francisella tularensis LVS, encoding the heat shock proteins chaperone-10 (Cpn10) and Cpn60, was sequenced and characterized, and the T-cell response of LVS-vaccinated individuals to the two proteins and the third major chaperone, Ft-DnaK, was assayed. The cpn10 and cpn60 genes were amplified by PCR with degenerate oligonucleotides derived from the N-terminal sequence of the two proteins. The sequence analysis revealed the expected two open reading frames, encoding proteins with estimated Mrs of 10,300 and 57,400. The deduced amino acid sequences closely resembled Cpn10 and Cpn60 proteins of other prokaryotes. The genes constituted a bicistronic operon, the cpn10 gene preceding the cpn60 gene. Upstream of the cpn10 gene, an inverted repeat and motifs similar to -35 and -10 sequences of sigma70-dependent but not of sigma32-dependent promoters of Escherichia coli were found. The inverted repeat of the operon resembled so-called hairpin loops identified in other characterized prokaryotic groE operons lacking sigma32-dependent promoters. Primer extension analysis disclosed one and the same transcription start, irrespective of the presence or absence of heat or oxidative stress. After separation of lysates of the F. tularensis LVS organism by two-dimensional gel electrophoresis, DnaK, Cpn60, and Cpn10 were extracted and used as antigens in T-cell tests. When compared to those from nonvaccinated individuals, T cells from individuals previously vaccinated with live F. tularensis LVS showed an increased proliferative response to DnaK and Cpn60 but not to Cpn10. The present data will facilitate further studies of the involvement of the heat shock proteins in protective immunity to tularemia.
Project description:In vitro refolding of pig mitochondrial malate dehydrogenase is investigated in the presence of Escherichia coli chaperonins cpn60 (groEL) and cpn10 (groES). When the enzyme is initially denatured with 3 M guanidinium chloride, chaperonin-assisted refolding is 100% efficient. C.d. spectroscopy reveals that malate dehydrogenase is almost unfolded in 3 M guanidinium chloride, suggesting that a state with little or no residual secondary structure is the optimal 'substrate' for chaperonin-assisted refolding. Malate dehydrogenase denatured to more highly structured states proves to refold less efficiently with chaperonin assistance. The enzyme is shown not to aggregate under the refolding conditions, so that losses in refolding efficiency result from irreversible misfolding. Evidence is advanced to suggest that the chaperonins are unable to rescue irreversibly misfolded malate dehydrogenase. A novel use is made of 100 K Centricon concentrators to study the binding of [14C]acetyl-labelled malate dehydrogenase to groEL by an ultrafiltration binding assay. Analysis of the data by Scatchard plot shows that acetyl-malate dehydrogenase, which has previously been extensively unfolded with guanidinium chloride, binds to groEL at a specific binding site(s). At saturation, one acetyl-malate dehydrogenase homodimer (two polypeptides) is shown to bind to each groEL homooligomer with a binding constant of approx. 10 nM.
Project description:BACKGROUND: Leishmania spp., in the course of their parasitic life cycle, encounter two vastly different environments: the gut of sandflies and the phagosomes of mammalian macrophages. During transmission into a mammal, the parasites are exposed to increased ambient temperature as well as to different carbon sources. Molecular chaperones or heat shock proteins are implicated in the necessary adaptations which involve the ordered differentiation from the flagellated, extracellular promastigote to the intracellular amastigote stage. RESULTS: Here, we show that the Leishmania donovani co-chaperonin, CPN10, is synthesised to a significantly increased concentration during in vitro differentiation to the amastigote stage. We show by fluorescence microscopy and by immunogold electron microscopy that, like its putative complex partner CPN60.2, CPN10 is localised to the single, tubular mitochondrion of the parasites and, moreover, that it co-precipitates with CPN60.2, the major mitochondrial chaperonin of Leishmania spp.. CONCLUSION: Our data indicate an increased requirement for CPN10 in the context of mitochondrial protein folding during or early in the mammalian stage of this pathogen. Moreover, they confirm the CPN60.2 as bona fide mitochondrial GroEL homologue in L. donovani and the postulated interaction of eukaryotic chaperonins, CPN60 and CPN10.
Project description:Chaperonin 60 (cpn60) and chaperonin 10 (cpn10) constitute the chaperonin system in prokaryotes, mitochondria, and chloroplasts. In Escherichia coli, these two chaperonins are also termed groEL and groES. We have used a functional assay to identify the groES homolog cpn10 in yeast mitochondria. When dimeric ribulose-1,5-bisphosphate carboxylase (Rubisco) is denatured and allowed to bind to yeast cpn60, subsequent refolding of Rubisco is strictly dependent upon yeast cpn10. The heterologous combination of cpn60 from E. coli plus yeast cpn10 is also functional. In contrast, yeast cpn60 plus E. coli cpn10 do not support refolding of Rubisco. In the presence of MgATP, yeast cpn60 and yeast cpn10 form a stable complex that can be isolated by gel filtration and that facilitates refolding of denatured Rubisco. Although the potassium-dependent ATPase activity of E. coli cpn60 can be inhibited by cpn10 from either E. coli or yeast, neither of these cpn10s inhibits the ATPase activity of yeast cpn60. Amino acid sequencing of yeast cpn10 reveals substantial similarity to the corresponding cpn10 proteins from rat mitochondria and prokaryotes.
Project description:The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.
Project description:Chloroplasts of higher plants contain a nuclear-encoded protein that is a functional homolog of the Escherichia coli chaperonin 10 (cpn10; also known as groES). In pea (Pisum sativum), chloroplast cpn10 was identified by its ability to (i) assist bacterial chaperonin 60 (cpn60; also known as groEL) in the ATP-dependent refolding of chemically denatured ribulose-1,5-bisphosphate carboxylase and (ii) form a stable complex with bacterial cpn60 in the presence of Mg.ATP. The subunit size of the pea protein is approximately 24 kDa--about twice the size of bacterial cpn10. A cDNA encoding a spinach (Spinacea oleracea) chloroplast cpn10 was isolated, sequenced, and expressed in vitro. The spinach protein is synthesized as a higher molecular mass precursor and has a typical chloroplast transit peptide. Surprisingly, however, attached to the transit peptide is a single protein, comprised of two distinct cpn10 molecules in tandem. Moreover, both halves of this "double" cpn10 are highly conserved at a number of residues that are present in all cpn10s that have been examined. Upon import into chloroplasts the spinach cpn10 precursor is processed to its mature form of approximately 24 kDa. N-terminal amino acid sequence analysis reveals that the mature pea and spinach cpn10 are identical at 13 of 21 residues.
Project description:Homologues of the chaperonins Cpn60 and Cpn10 have been purified from the Gram-positive cellulolytic thermophile Clostridium thermocellum. The Cpn60 protein was purified by ATP-affinity chromatography and the Cpn10 protein was purified by gel-filtration, ion-exchange and hydrophobic interaction chromatographies. The identities of the proteins were confirmed by N-terminal sequence analysis and antigenic cross-reactivity. The Cpn60 homologue is a weak, thermostable ATPase (t1/2 at 70 decrees C more than 90 min) with optimum activity (Kcat 0.07 S-1) between 60 degrees C and 70 degrees C. The ATPase activity of the authentic Cpn60 was inhibited by Escherichia coli GroES. The catalytic properties of a recombinant C. thermocellum Cpn60 purified from a GST-Cpn60 fusion protein expressed in E. coli [Ciruela (1995) Ph.D. Thesis, University of Kent] were identical with those of the authentic C. thermocellum Cpn60. Gel-filtration studies show that at room temperature the Cpn60 migrates mainly as a heptamer. Electron microscopy confirms the presence of complexes showing 7-fold rotational symmetry and also reveals a small number of particles that seem to be tetradecamers with a similar structure to E. coli GroEL complexes.
Project description:Heat shock proteins (HSP) might be useful as biomarkers for bipolar disorder (BD) which would be clinically valuable since no reliable biomarker for BD has so far been identified. The purpose of this study was to assess the heat shock proteins CPN10, CPN60, and CPN70 as potential biomarkers of BD.The study included 100 BD patients recruited from a hospital during 2012 and 2013. The study also included 94 healthy controls. Among the BD patients, 33 had abnormal hypothalamic-pituitary-adrenal (HPA) axis activity. Blood samples were obtained from the patients and controls. The chemiluminescence method, mass spectrometry, and flow cytometry were used for analysis.The BD patients compared with the controls had a significantly lower level of CPN10 and significantly higher levels of CPN60 and CPN70. The BD patients with abnormal HPA axis activity had a significantly lower level of CPN60 compared with the normal HPA axis activity group of BD patients. The CPN60 level significantly inversely correlated with adrenocorticotropic hormone (ACTH) level in patients with bipolar depression and in patients with bipolar hypomania, and CPN70 significantly correlated with ACTH level in patients with bipolar depression and hypomania.Our findings suggest that the heat shock proteins CPN10, CPN60, and CPN70 might have potential as biomarkers for BD and CPN60 blood level might distinguish patients with abnormal HPA axis activity from those with normal HPA axis activity.
Project description:The crystal structure of Mycobacterium tuberculosis chaperonin 10 (cpn10(Mt)) has been determined to a resolution of 2.8 A. Two dome-shaped cpn10(Mt) heptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure. The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 A perpendicular to and 60 A along the sevenfold axis. Tetradecameric cpn10(Mt) has also been observed in solution by dynamic light scattering. Through its base loop sequence cpn10(Mt) is known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity. Superimposition of the cpn10(Mt) sequences 26 to 32 and 66 to 72 and E. coli GroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences. The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo. Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer. This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.