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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.
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PMID:Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. 136 Nov 69

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.
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PMID:Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides. 167 80

Many proteins in the cell require assistance from molecular chaperones at stages in their life cycles in order to attain correctly folded states and functional conformations during protein synthesis or during recovery from denatured states. A recently discovered molecular chaperone, which is abundant in the eukaryotic cytosol and is called the chaperonin containing TCP-1 (CCT), has been shown to assist the folding of some proteins in cytosol. This chaperone is a member of the chaperonin family which includes GroEL, 60-kDa heat shock protein (Hsp60), Rubisco subunit binding protein (RBP) and thermophilic factor 55 (TF55), but is distinct from the other members in several respects. Presently the most intriguing feature is the hetero-oligomeric nature of the CCT; at least eight subunit species which are encoded by independent and highly diverged genes are known. These genes are calculated to have diverged around the starting point of the eukaryotic lineage and they are maintained in all eukaryotes investigated, suggesting a specific function for each subunit species. The amino acid sequences of these subunits share approximately 30% identity and have some highly conserved motifs probably responsible for ATPase function, suggesting this function is common to all subunits. Thus, each subunit is thought to have both specific and common functions. These observations, in conjunction with biochemical and genetic analysis, suggest that CCT functions as a very complex machinery for protein folding in the eukaryotic cell and that its chaperone activity may be essential for the folding and assembly of various newly synthesized polypeptides. This complex behaviour of CCT may have evolved to cope with the folding and assembly of certain highly evolved proteins in eukaryotic cells.
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PMID:The chaperonin containing t-complex polypeptide 1 (TCP-1). Multisubunit machinery assisting in protein folding and assembly in the eukaryotic cytosol. 760 Nov 14

All major classes of protein chaperones, including DnaK (the Hsp70 eukaryotic equivalent) and GroEL (the Hsp60 eukaryotic equivalent) have been found in Escherichia coli. Molecular chaperones enhance the yields of correctly folded polypeptides by preventing aggregation and even by disaggregating certain protein aggregates. Previously, we identified the ClpX heat-shock protein of E. coli because it enables the ClpP catalytic protease to degrade the bacteriophage lambda O replication protein. Here we report that ClpX alone possesses all the properties expected of a molecular chaperone protein. Specifically, it can protect the lambda O protein from heat-induced aggregation, disaggregate preformed lambda O aggregates, and even promote efficient binding of lambda O to its DNA recognition sequence. A lambda O-ClpX specific protein-protein interaction can be detected either by a modified ELISA assay or through the stimulation of ClpX's weak ATPase activity by lambda O. Unlike the behaviour of the major DnaK and GroEL chaperones, ClpX requires the presence of ATP or its non-hydrolysable analogue ATP-gamma-S for efficient interaction with other proteins including the protection of lambda O from aggregation. However, ClpX's ability to disaggregate lambda O aggregates requires hydrolysable ATP. We propose that the ClpX protein is a bona fide chaperone, whose biological role includes the maintenance of certain polypeptides in a form competent for proteolysis by the ClpP protease. Furthermore, our results suggest that the ClpX protein also performs typical chaperone protein functions independent of ClpP.
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PMID:The ClpX heat-shock protein of Escherichia coli, the ATP-dependent substrate specificity component of the ClpP-ClpX protease, is a novel molecular chaperone. 774 94

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.
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PMID:Identification and functional analysis of chaperonin 10, the groES homolog from yeast mitochondria. 790 76

GroEL, an Escherichia coli homolog of the heat shock protein 60 family of molecular chaperonins, has been implicated as a target of T cell-mediated immune responses in a broad spectrum of infections. In order to produce large quantities of native protein for raising and stimulating GroEL specific T cell lines, we have developed a simple and rapid two-step protocol for purifying native E. coli GroEL heat shock (or stress) protein which takes advantage of the inherent structural and functional properties of the protein. Based on a combination of gel exclusion chromatography, ATPase activity assay, isoelectric focusing, and circular dichroism analyses we conclude that our purification process yields native tetradecameric GroEL.
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PMID:A simple and rapid method for the purification of GroEL, an Escherichia coli homolog of the heat shock protein 60 family of molecular chaperonins. 790 92

Protein folding in vivo is mediated by helper proteins, the molecular chaperones, of which Hsp60 and its Escherichia coli variant GroEL are some of the best characterized. GroEL is an oligomeric protein with 14 subunits each of M(r) 60K, which possesses weak, co-operative ATPase activity and high plasticity. GroEL seems to interact with non-native proteins, binding one or two molecules per 14-mer in a 'central cavity', but little is known about the conformational state of the bound polypeptides. Here we use nuclear magnetic resonance techniques to show that the interaction of the small protein cyclophilin with GroEL is reversible by temperature changes, and all amide protons in GroEL-bound cyclophilin are exchanged with the solvent, although this exchange does not occur in free cyclophilin. The complete secondary structure of cyclophilin must be disrupted when bound to GroEL.
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PMID:Destabilization of the complete protein secondary structure on binding to the chaperone GroEL. 790 13

Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2-propanol or poly(ethylene glycol) with MgCl2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 +/- 0.4 Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein-folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.
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PMID:Purification and characterization of chaperonin 60 and chaperonin 10 from the anaerobic thermophile Thermoanaerobacter brockii. 791 71

Decreased intracellular levels of FtsH, a membrane-bound ATPase, led to retardation of growth and protein export, as well as to an abnormal translocation of alkaline phosphatase that had been attached to a cytoplasmic domain of a multispanning membrane protein, SecY. The last phenotype is designated Std (stop transfer defective). In this study, we examined the effects of overproduction of some molecular chaperones on the phenotypes of ftsH mutants. The growth retardation was partially suppressed by overproduction of GroEL/GroES (Hsp60/Hsp10) or HtpG (Hsp90), although these chaperones could not totally substitute for FtsH. Overproduction of HtpG specifically alleviated the Std phenotype, while that of GroEL/GroES alleviated the protein export defect of ftsH mutants. These results suggest that FtsH functions can be somehow compensated for when the cellular concentrations of some molecular chaperones increase.
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PMID:Suppression of ftsH mutant phenotypes by overproduction of molecular chaperones. 857 50

Na+,K(+)-ATPase activity and its alpha 1 subunit protein and mRNA in kidney cortex were monitored in rats developing Fanconi syndrome after the administration of maleate. Na+,K(+)-ATPase activity was significantly lower than in saline-injected controls, although this was partially mediated by a general, non-specific decrease in the cortex protein content. 2. The low activity of the sodium pump correlated with low abundance of alpha 1 subunit mRNA and protein levels. Hsp60 protein levels were also decreased in kidney cortex from maleate-treated rats. 3. Kidney cortex brush-border membrane vesicles from maleate-treated rats showed a marked decrease in Na(+)-dependent alanine and glucose transport, which was not dependent on the Na(+)-transmembrane gradient itself, a finding which is consistent with a more stable effect at the plasma membrane level. 4. The effect of maleate may be partially non-specific and involve a great variety of proteins, but seems to be restricted to selected tissues because alpha 1 subunit Na+,K(+)-ATPase and hsp60 protein amounts were not significantly modified in livers from rats developing Fanconi syndrome. 5. These results show that maleate administration induces a low activity of selected concentrative transport systems and a decrease in Na+,K(+)-ATPase activity and expression. The combination of both effects may explain the increased excretion of most organic solutes present in rats developing Fanconi syndrome.
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PMID:Na+,K(+)-ATPase expression in maleic-acid-induced Fanconi syndrome in rats. 909 4


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