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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)

Chaperonin-facilitated folding of proteins involves two partial reactions. The first partial reaction, the formation of stable binary complexes between chaperonin-60 and non-native states of the target protein, is common to the chaperonin-facilitated folding of all target proteins investigated to date. The structural basis for this interaction is not presently understood. The second partial reaction, the dissociation of the target protein in a form committed to the native state, appears to proceed by a variety of mechanisms, dependent upon the nature of the target protein in question. Those target proteins (e.g. rubisco, rhodanese, citrate synthase) which require the presence of chaperonin-10, also appear to require the hydrolysis of ATP to bring about the dissociation of the target protein from chaperonin-60. With one exception (pre-beta-lactamase) those target proteins which do not require the presence of chaperonin-10 to be released from chaperonin-60, also do not require the hydrolysis of ATP, since non-hydrolysable analogues of ATP support the release of the target protein in a state committed to the native state. The question of whether or not chaperonin-facilitated folding constitutes a catalysed event is addressed.
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PMID:Chaperonins and protein folding: unity and disunity of mechanisms. 809 34

Ligand-induced conformational changes of GroEL alone and with bound rhodanese, citrate synthase, or dihydrofolate reductase were studied by limited proteolysis. Similar digestion patterns of GroEL, with or without bound substrate polypeptide, were obtained in the absence and presence of the chaperonin ligands, K+, Mg2+, or ATP. The rates of formation and degradation of the six produced proteolytic fragments were significantly different, however. Strikingly, only with Mg2+/ATP or K+/Mg2+/ATP an additional fragment of approximately 25 kDa was generated during digestion of GroEL alone or with bound rhodanese or dihydrofolate reductase, but not with bound citrate synthase. Most of the trypsin-sensitive sites in GroEL were localized in the flexible apical domain, which contains the putative polypeptide-binding region. Our data indicate that subtle structural changes in the trypsin-sensitive regions of GroEL occur as a result of the binding of the chaperonin ligands. However, these structural changes are influenced by the GroEL substrate polypeptides.
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PMID:Ligand-induced conformational changes of GroEL are dependent on the bound substrate polypeptide. 866 87

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions.
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PMID:The Hsc66-Hsc20 chaperone system in Escherichia coli: chaperone activity and interactions with the DnaK-DnaJ-grpE system. 985 6

Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis.
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PMID:Nucleolar protein B23 has molecular chaperone activities. 1021 37

IscU, a NifU-like Fe/S-escort protein, binds to and stimulates the ATPase activity of Hsc66, a hsp70-type molecular chaperone. We present evidence that stimulation arises from interactions of IscU with the substrate-binding site of Hsc66. IscU inhibited the ability of Hsc66 to suppress the aggregation of the denatured model substrate proteins rhodanese and citrate synthase, and calorimetric and surface plasmon resonance measurements showed that ATP destabilizes Hsc66.IscU complexes in a manner expected for hsp70-substrate complexes. Studies on the interaction of IscU with Hsc66 truncation mutants further showed that IscU does not bind the isolated ATPase domain of Hsc66 but does bind and stimulate a mutant containing the ATPase domain and substrate binding beta-sandwich subdomain. These results support a role for IscU as a substrate for Hsc66 and suggest a specialized function for Hsc66 in the assembly, stabilization, or transfer of Fe/S clusters formed on IscU.
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PMID:The Fe/S assembly protein IscU behaves as a substrate for the molecular chaperone Hsc66 from Escherichia coli. 1105 47

Human P5 (hP5) was expressed in the Escherichia coli pET system and purified by sequential Ni(2+)-chelating resin column chromatography. Characterization of purified hP5 indicated that it has both isomerase and chaperone activities, but both activities are lower than those of human protein disulfide isomerase (PDI). Moreover, hP5 was observed to have peptide-binding ability, and its chaperone activity was confirmed with rhodanese and citrate synthase as substrates, but not with D-glyceraldehyde-3-phosphate dehydrogenase, showing that hP5 has substrate specificity with respect to chaperone activity. Mutation of two thioredoxin-related motifs in hP5 revealed that the first motif is more important than the second for isomerase activity and that the first cysteine in each motif is necessary for isomerase activity. Since thioredoxin motif mutants lacking isomerase activity retain chaperone activity with the substrate citrate synthase, the isomerase and chaperone activities of hP5 are probably independent, as was shown for PDI.
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PMID:Functional analysis of human P5, a protein disulfide isomerase homologue. 1220 15

The non-ionic detergent Brij 58P is recommended as a stabilizing agent for protein storage; for example, the aggregation-prone chaperone DnaJ can be maintained in solution by low concentrations of Brij 58P. During protein folding studies with alpha-glucosidase, rhodanese and citrate synthase as model proteins, we discovered that the low concentrations of Brij 58P usually added with purified DnaJ to renaturation samples are sufficient to mimic chaperone effects with respect to prevention of protein aggregation. Furthermore, addition of Brij 58P to refolding alpha-glucosidase and citrate synthase enhanced the yield of refolded protein by a factor of two. Thus, Brij 58P can mimic chaperone effects and care should be taken when the substance is used to stabilize chaperone preparations.
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PMID:The non-ionic detergent Brij 58P mimics chaperone effects. 1245

Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.
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PMID:Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23. 1251 51

We report the characterization of the first chaperonin (Mm-cpn) from a mesophilic archaeon, Methanococcus maripaludis. The single gene was cloned from genomic DNA and expressed in Escherichia coli to produce a recombinant protein of 543 amino acids. In contrast with other known archaeal chaperonins, Mm-cpn is fully functional in all respects under physiological conditions of 37 degrees C. The complex has Mg(2+)-dependent ATPase activity and can prevent the aggregation of citrate synthase. It promotes a high-yield refolding of guanidinium-chloride-denatured rhodanese in a nucleotide-dependent manner. ATP binding is sufficient to effect folding, but ATP hydrolysis is not essential.
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PMID:Nucleotide-dependent protein folding in the type II chaperonin from the mesophilic archaeon Methanococcus maripaludis. 1262

The ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding. No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, alpha-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding. The two sHsps differ in their modes of activation. In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding. Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins. For all substrates investigated, complexes of defined size and shape were formed. Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes. The first substrate protein bound seems to determine the complex morphology. Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals.
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PMID:Analysis of the interaction of small heat shock proteins with unfolding proteins. 1263 95


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