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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The folding of many newly synthesized proteins in the cell depends on a set of conserved proteins known as molecular chaperones. These prevent the formation of misfolded protein structures, both under normal conditions and when cells are exposed to stresses such as high temperature. Significant progress has been made in the understanding of the ATP-dependent mechanisms used by the Hsp70 and chaperonin families of molecular chaperones, which can cooperate to assist in folding new polypeptide chains.
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PMID:Molecular chaperones in cellular protein folding. 863 92

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

The Escherichia coli chaperonins GroEL and GroES facilitate the refolding of polypeptide chains in an ATP hydrolysis-dependent reaction. The elementary steps in the binding and release of polypeptide substrates to GroEL were investigated in surface plasmon resonance studies to measure the rates of binding and dissociation of a normative variant of subtilisin. The rate constants determined for GroEL association with and dissociation from this variant yielded a micromolar dissociation constant, in agreement with independent calorimetric estimates. The rate of GroEL dissociation from the nonnative chain was increased significantly in the presence of 5'-adenylylimidodiphosphate (AMP-PNP), ADP, and ATP, yielding maximal values between 0.04 and 0.22 s(-1). The sigmoidal dependence of the dissociation rate on the concentration of AMP-PNP and ADP indicated that polypeptide dissociation is limited by a concerted conformational change that occurs after nucleotide binding. The dependence of the rate of release on ATP exhibited two sigmoidal transitions attributable to nucleotide binding to the distal and proximal toroid of a GroEL-polypeptide chain complex. The addition of GroES resulted in a marked increase in the rate of nonnative polypeptide release from GroEL, indicating that the cochaperonin binds more rapidly than the dissociation of polypeptides. These data demonstrate the importance of nucleotide binding-promoted concerted conformational changes for the release of chains from GroEL, which correlate with the sigmoidal hydrolysis of ATP by the chaperonin. The implications of these findings are discussed in terms of a working hypothesis for a single cycle of chaperonin action.
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PMID:Nucleotide binding-promoted conformational changes release a nonnative polypeptide from the Escherichia coli chaperonin GroEL. 870 Aug 70

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the chaperonin containing TCP-1 (CCT) and, in the case of alpha- and beta-tubulin additional protein cofactors. We recently identified one of these cofactors, termed cofactor A, that is required for the proper folding of the beta-tubulin chain [Gao et al. (1994) J. Cell. Biol. 125, 989-996]. We show here that cofactor A, a monomeric protein that has no measurable affinity for nucleotides, is a highly conserved protein among vertebrates. Its NH2-terminal region is essential for the structural integrity of the protein and consequently for its activity. We demonstrate that cofactor A does not interact with CCT nor does it affect the intrinsic ATPase activity of CCT, alone or in the presence of different target proteins. Thus, unlike GroES, cofactor A does not modulate or coordinate ATP hydrolysis. It does not act as a nucleotide exchange factor or a catalyst in tubulin folding. Rather, we demonstrate that cofactor A participates in the tubulin folding process by interacting with a folding intermediate of beta-tubulin that is released from CCT. Our data imply that cofactor A is a chaperone involved in tubulin folding.
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PMID:Cofactor A is a molecular chaperone required for beta-tubulin folding: functional and structural characterization. 875 98

A homologue of the chaperonin protein of the HSP60 family has not been shown so far in Drosophila. Using an antibody specific to HSP60 family protein in Western blotting and immunocytochemistry, we showed that a 64-kDa polypeptide, homologous to the HSP60, is constitutively present in all tissues of Drosophila melanogaster throughout the life cycle from the freshly laid egg to all embryonic, larval and adult stages. A 64-kDa polypeptide reacting with the same antibody in Western blots is present in all species of Drosophila examined. Using Western blotting in conjunction with 35S-methionine labeling of newly synthesized proteins and immuno-precipitation of the labeled proteins with HSP60-specific antibody, it was shown that synthesis of the 64-kDa homologue of HSP60 is appreciably increased by heat shock only in the Malpighian tubules, which are already known to lack the common HSPs.
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PMID:Synthesis of a ubiquitously present new HSP60 family protein is enhanced by heat shock only in the Malpighian tubules of Drosophila. 877 44

Protein folding by the double-ring chaperonin GroEL is initiated in cis ternary complexes, in which polypeptide is sequestered in the central channel of a GroEL ring, capped by the co-chaperonin GroES. The cis ternary complex is dissociated (half-life of approximately 15 s) by trans-sided ATP hydrolysis, which triggers release of GroES. For the substrate protein rhodanese, only approximately 15% of cis-localized molecules attain their native form before hydrolysis. A major question concerning the GroEL mechanism is whether both native and non-native forms are released from the cis complex. Here we address this question using a 'cis-only' mixed-ring GroEL complex that binds polypeptide and GroES on only one of its two rings. This complex mediates refolding of rhodanese but, as with wild-type GroEL, renaturation is quenched by addition of mutant GroEL 'traps', which bind but do not release polypeptide substrate. This indicates that non-native forms are released from the cis complex. Quenching of refolding by traps was also observed under physiological conditions, both in undiluted Xenopus oocyte extract and in intact oocytes. We conclude that release of non-native forms from GroEL in vivo allows a kinetic partitioning among various chaperones and proteolytic components, which determines both the conformation and lifetime of a protein.
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PMID:Release of both native and non-native proteins from a cis-only GroEL ternary complex. 877 22

In the course of removing a contaminant from preparations of aminoacyl-tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin-agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino-terminal sequencing, and ATP-dependent refolding of rhodanese and actin. Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the "characteristic" ring and four-stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate-containing views are observed. The particle structure is strikingly resistant to physical forces (long-term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KCl or LiCl), and pH changes (9-6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KCl or deoxycholate reduces TRiC folding activity.
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PMID:Novel isolation method and structural stability of a eukaryotic chaperonin: the TCP-1 ring complex from rabbit reticulocytes. 881 69

We have identified a gene encoding the 60 kDa heat shock protein (hsp60) from a Plasmodium falciparum blood stage cDNA library. The deduced protein sequence encodes for a polypeptide of 577 amino acids with a calculated molecular weight of 62158 Da. The primary structure of P. falciparum hsp60 contains a putative mitochondrial targeting peptide at its amino-terminus and a GGM motif at its carboxyl-terminus. The overall structure exhibits strong conservation (approximately 50%) to the hsp60 from human and other eukaryotes, but only low homology (< 30%) to a recently reported P. falciparum chaperonin 60 gene. The P. falciparum hsp60 gene is located on chromosome 10. During heat shock, the level of hsp60 transcript in blood stage parasites increases significantly and its accumulation correlates with the duration of the induction.
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PMID:Cloning of a Plasmodium falciparum gene related to the human 60-kDa heat shock protein. 884 68

Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities. Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex. The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins. When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra. Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.
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PMID:Interplay of structure and disorder in cochaperonin mobile loops. 887 86

It has been believed that when GroEL binds to GroES its apical domain moves upward and outward. To inhibit this "opening" movement, its equatorial and apical domains were cross-linked through a disulfide bond between mutationally introduced cysteine residues at the positions of Asp-83 and Lys-327. To avoid possible undesired cross-linking, we at first prepared a mutant GroEL (GroELNC; Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser) in which all cysteine residues in wild-type GroEL were replaced by serine residues. GroELNC was fully functional as a chaperonin. We then introduced the above two point mutations into GroELNC to generate a mutant (GroELAEX; Cys-138 --> Ser, Cys-458 --> Ser, Cys-519 --> Ser and Asp-83 --> Cys, Lys-327 --> Cys). Oxidized GroELAEX, which is locked in a "closed" conformation by an interdomain disulfide bond, can bind 6-7 mol of ATP, which remain bound without hydrolysis. This ATP-bound, oxidized GroELAEX can bind the stably nonnative substrate protein isopropylmalate dehydrogenase, whereas the nucleotide-free oxidized GroELAEX binds it with a weaker affinity. However, oxidized GroELAEX fails to process further reaction steps such as ATP hydrolysis, binding of GroES, dissociation of substrate protein from GroEL, and facilitating protein folding. When disulfide bonds in oxidized GroELAEX are reduced, GroELAEX exerts the ability to process all the reactions just as GroELNC and wild-type GroEL. Indications from these results are: hydrolysis of ATP may require opening movement of the apical domain; GroES binds to an open form of GroEL; and substrate polypeptide is released from GroEL coupled with either ATP hydrolysis or opening movement of the apical domain.
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PMID:GroEL locked in a closed conformation by an interdomain cross-link can bind ATP and polypeptide but cannot process further reaction steps. 891 Apr 40


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