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Biomedical subjects

F U Hartl

Publications and source records attributed to F U Hartl.

At least 73 records · Page 4Linked to original sources
PubMed1995

Asymmetrical interaction of GroEL and GroES in the ATPase cycle of assisted protein folding.

The chaperonins GroEL and GroES of Escherichia coli facilitate protein folding in an adenosine triphosphate (ATP)-dependent reaction cycle. The kinetic parameters for the formation and dissociation of GroEL-GroES complexes were analyzed by surface plasmon resonance. Association of GroES and subsequent ATP hydrolysis in the interacting GroEL toroid resulted in the formation of a stable GroEL:ADP:GroES complex. The complex dissociated as a result of ATP hydrolysis in the opposite GroEL toroid, without formation of a symmetrical GroEL:(GroES)2 intermediate. Dissociation was accelerated by the addition of unfolded polypeptide. Thus, the functional chaperonin unit is an asymmetrical GroEL:GroES complex, and substrate protein plays an active role in modulating the chaperonin reaction cycle.

Adenosine Diphosphate↗
PubMed1995

Principles of chaperone-mediated protein folding.

The recent discovery of molecular chaperones and their functions has changed dramatically our view of the processes underlying the folding of proteins in vivo. Rather than folding spontaneously, most newly synthesized polypeptide chains seem to acquire their native conformations in a reaction mediated by chaperone proteins. Different classes of molecular chaperones, such as the members of the Hsp70 and Hsp60 families of heat-shock proteins, cooperate in a coordinated pathway of cellular protein folding.

Chaperonin 60↗
PubMed1995

The thermosome of Thermoplasma acidophilum and its relationship to the eukaryotic chaperonin TRiC.

A high molecular-mass protein complex from the archaebacterium Thermoplasma acidophilum, referred to here as the 'thermosome', is built from two subunits (M(r) 58 and 60). The thermosome has been purified to homogeneity. The molecular mass of the native complex was determined to be 1061 +/- 30 Da by scanning transmission electron microscopy. It shows a weak ATPase activity and is able to bind denatured polypeptides. Averages obtained from electron micrographs of negatively stained molecules in the end-on and side-on orientations, respectively, were compared with those of the t-complex polypeptide 1 ring complex (TRiC), isolated from bovine testes. Both molecules consist of two stacked pseudo eightfold symmetric rings which build up a cylindrical particle with a large cavity in the center. Sequence alignments of peptides generated from both subunits of the thermosome and different subunits of TRiC reveal a high partial similarity to each other and to the archaebacterial chaperonin thermophilic factor 55 from Sulfolobus shibatae as well as to eukaryotic TCP1 proteins. These striking structural similarities confirm the proposition that all these molecules belong to a single protein family which is structurally and functionally related to the GroEL class of molecular chaperones.

Adenosine Triphosphatases↗
PubMed1995

Molecular chaperones in cellular protein folding.

Protein folding in the cell requires molecular chaperones. The chaperone proteins of the hsp70 and hsp60 (chaperonin) classes stabilize unfolded or partially folded polypeptides, thereby preventing aggregation, and mediate folding to the native state in ATP-dependent reactions. Recent advances include a more detailed understanding of the mechanistic principles of hsp70 and hsp60 action, the solution of the crystal structure of the chaperonin GroEL, and the definition of pathways of chaperone-mediated protein folding.

Chaperonins↗
PubMed1995

Binding of defined regions of a polypeptide to GroEL and its implications for chaperonin-mediated protein folding.

Unfolded rhodanese in a complex with the chaperonin GroEL was subjected to limited proteolysis. Sequence analysis indentified a GroEL-bound fragment of approximately 11,000 M(r) and a well defined fragment of approximately 7,000 M(r) from the two homologous domains of rhodanese. The shorter segment contains one hydrophobic and one amphiphilic alpha-helix mapping to the domain interface while the other fragment contains the homologous regions and an additional hydrophobic helix. Our results suggest a mechanism for the GroEL-mediated folding of rhodanese in which the domain-forming regions of the polypeptide are kept apart and are then released, perhaps sequentially, resulting in correct folding.

Amino Acid Sequence↗
PubMed1995

The role of molecular chaperones in protein folding.

Folding of newly synthesized polypeptides in the crowded cellular environment requires the assistance of so-called molecular chaperone proteins. Chaperones of the Hsp70 class and their partner proteins interact with nascent polypeptide chains on ribosomes and prevent their premature (mis)folding at least until a domain capable of forming a stable structure is synthesized. For many proteins, completion of folding requires the subsequent interaction with one of the large oligomeric ring-shaped proteins of the chaperonin family, which is composed of the GroEL-like proteins in eubacteria, mitochondria, and chloroplasts, and the TRiC family in eukaryotic cytosol and archaea. These proteins bind partially folded polypeptide in their central cavity and promote folding by ATP-dependent cycles of release and rebinding. In these reactions, molecular chaperones interact predominantly with the hydrophobic surfaces exposed by nonnative polypeptides, thereby preventing incorrect folding and aggregation.

Animals↗
PubMed1995

Identification of GroEL as a constituent of an mRNA-protection complex in Escherichia coli.

An RNA-binding activity has been identified in Escherichia coli that provides physical protection of RNA against ribonucleases in an ATP- and Mg(2+)-dependent manner. This binding activity is stimulated under growth conditions known to cause a decrease in the rate of mRNA decay. RNA protection is mediated by a protein complex that contains a modified form of the chaperonin GroEL as an indispensable constituent. These results suggest a new role for GroEL as an RNA chaperone.

Adenosine Diphosphate↗
PubMed1994

Conformation of GroEL-bound alpha-lactalbumin probed by mass spectrometry.

The conformation of a three-disulphide derivative of bovine alpha-lactalbumin bound to the molecular chaperone GroEL has been investigated by monitoring directly its hydrogen exchange kinetics using electrospray ionization mass spectrometry. The bound protein is weakly protected from exchange to an extent closely similar to that of an uncomplexed molten globule state of the three-disulphide protein. Binding to GroEL in this system appears to involve relatively disordered partly folded states resembling intermediates formed in the very early stages of kinetic folding of many proteins in vitro.

Animals↗
PubMed1994

The ATP hydrolysis-dependent reaction cycle of the Escherichia coli Hsp70 system DnaK, DnaJ, and GrpE.

Molecular chaperones of the Hsp70 class bind unfolded polypeptide chains and are thought to be involved in the cellular folding pathway of many proteins. DnaK, the Hsp70 protein of Escherichia coli, is regulated by the chaperone protein DnaJ and the cofactor GrpE. To gain a biologically relevant understanding of the mechanism of Hsp70 action, we have analyzed a model reaction in which DnaK, DnaJ, and GrpE mediate the folding of denatured firefly luciferase. The binding and release of substrate protein for folding involves the following ATP hydrolysis-dependent cycle: (i) unfolded luciferase binds initially to DnaJ; (ii) upon interaction with luciferase-DnaJ, DnaK hydrolyzes its bound ATP, resulting in the formation of a stable luciferase-DnaK-DnaJ complex; (iii) GrpE releases ADP from DnaK; and (iv) ATP binding to DnaK triggers the release of substrate protein, thus completing the reaction cycle. A single cycle of binding and release leads to folding of only a fraction of luciferase molecules. Several rounds of ATP-dependent interaction with DnaK and DnaJ are required for fully efficient folding.

Adenosine Triphosphatases↗
PubMed1994

Tcp20, a subunit of the eukaryotic TRiC chaperonin from humans and yeast.

Members of the Hsp60 chaperonin family, such as Escherichia coli GroEL/S and the eukaryotic cytosolic chaperonin complex, TRiC (TCP ring complex), are double toroid complexes capable of assisting the folding of proteins in vitro in an ATP-dependent fashion. TRiC differs from the GroEL chaperonin in that it has a hetero rather than homo-oligomeric subunit composition and lacks a GroES-like regulatory cofactor. We have established greater than 57% identity between a protein encoded by the TCP20 gene from a human cDNA library and the newly identified protein encoded by the TCP20 gene located on the right arm of chromosome IV of the yeast Saccharomyces cerevisiae. These Tcp20 proteins showed approximately 30% identity to Tcp1, a known subunit of TRiC. Gel filtration, followed by Western analysis of purified bovine testis TRiC with a Tcp20-specific antibody, indicated that Tcp20 is a subunit of the hetero-oligomeric TRiC. Gene disruption experiments showed that TCP20, like TCP1, is an essential gene in yeast, consistent with the view that TRiC is required for folding of key proteins. The amino acid sequence similarities and the derived evolutionary relationships established that the human and yeast Tcp20 proteins represent members of a new family of subunits of TRiC chaperonins.

Amino Acid Sequence↗
PubMed1994

Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones.

The folding of polypeptides emerging from ribosomes was analysed in a mammalian translation system using firefly luciferase as a model protein. The growing polypeptide interacts with a specific set of molecular chaperones, including Hsp70, the DnaJ homologue Hsp40 and the chaperonin TRiC. The ordered assembly of these components on the nascent chain forms a high molecular mass complex that allows the cotranslational formation of protein domains and the completion of folding once the chain is released from the ribosome.

Adenosine Triphosphate↗
PubMed1994

Conformational specificity of the chaperonin GroEL for the compact folding intermediates of alpha-lactalbumin.

The chaperonin GroEL binds unfolded polypeptides, preventing aggregation, and then mediates their folding in an ATP-dependent process. To understand the structural features in non-native polypeptides recognized by GroEL, we have used alpha-lactalbumin (alpha LA) as a model substrate. alpha LA (14.2 kDa) is stabilized by four disulfide bonds and a bound Ca2+ ion, offering the possibility of trapping partially folded disulfide intermediates between the native and the fully unfolded state. The conformers of alpha LA with high affinity for GroEL are compact, containing up to three disulfide bonds, and have significant secondary structure, but lack stable tertiary structure and expose hydrophobic surfaces. Complex formation requires almost the complete alpha LA sequence and is strongly dependent on salts that stabilize hydrophobic interactions. Unfolding of alpha LA to an extended state as well as the burial of hydrophobic surface upon formation of ordered tertiary structure prevent the binding to GroEL. Interestingly, GroEL interacts only with a specific subset of the many partially folded disulfide intermediates of alpha LA and thus may influence in vitro the kinetics of the folding pathways that lead to disulfide bonds with native combinations. We conclude that the chaperonin interacts with the hydrophobic surfaces exposed by proteins in a flexible compact intermediate or molten globule state.

Animals↗
PubMed1994

Molecular chaperones in cellular protein folding.

The discovery of 'molecular chaperones' has dramatically changed our concept of cellular protein folding. Rather than folding spontaneously, most newly synthesized polypeptide chains seem to acquire their native conformation in a reaction mediated by these versatile helper proteins. Understanding the structure and function of molecular chaperones is likely to yield useful applications for medicine and biotechnology in the future.

Bacteria↗
PubMed1994

Post-translational protein import and folding.

Significant advances have been made over the past year in analyzing the membrane machineries for the post-translational export of proteins in bacteria and for the import of proteins into mitochondria. Another important development is the identification in mitochondria of homologs of the bacterial heat-shock proteins DnaJ and GrpE, which function together with Hsp70 in membrane translocation and folding of imported proteins. A number of gene products involved in peroxisomal protein uptake have been identified, which are now awaiting biochemical analysis.

Animals↗
PubMed1994

Molecular chaperones in protein folding: the art of avoiding sticky situations.

Molecular chaperones are a class of proteins that interact with the non-native conformations of other proteins. The major role of chaperones of the Hsp70 and Hsp60 families is to prevent aggregation of newly synthesized polypeptides and then to mediate their folding to the native state. As a result of functional studies of these proteins, there has been a revision of the long-held view that protein folding in the cell is a spontaneous process.

Animals↗
PubMed1994

Role of the chaperonin cofactor Hsp10 in protein folding and sorting in yeast mitochondria.

Protein folding in mitochondria is mediated by the chaperonin Hsp60, the homologue of E. coli GroEL. Mitochondria also contain a homologue of the cochaperonin GroES, called Hsp10, which is a functional regulator of the chaperonin. To define the in vivo role of the co-chaperonin, we have used the genetic and biochemical potential of the yeast S. cerevisiae. The HSP10 gene was cloned and sequenced and temperature-sensitive lethal hsp10 mutants were generated. Our results identify Hsp10 as an essential component of the mitochondrial protein folding apparatus, participating in various aspects of Hsp60 function. Hsp10 is required for the folding and assembly of proteins imported into the matrix compartment, and is involved in the sorting of certain proteins, such as the Rieske Fe/S protein, passing through the matrix en route to the intermembrane space. The folding of the precursor of cytosolic dihydrofolate reductase (DHFR), imported into mitochondria as a fusion protein, is apparently independent of Hsp10 function consistent with observations made for the chaperonin-mediated folding of DHFR in vitro. The temperature-sensitive mutations in Hsp10 map to a domain (residues 25-40) that corresponds to a previously identified mobile loop region of bacterial GroES and result in a reduced binding affinity of hsp10 for the chaperonin at the non-permissive temperature.

Amino Acid Sequence↗
PubMed1994

Topology of the morphological domains of the chaperonin GroEL visualized by immuno-electron microscopy.

Electron microscopy of the tetradecameric double-ring complex of GroEL reveals a four-layered structure, indicating that the 58 kDa subunits are composed of two major morphological domains. We have used immuno-electron microscopy to assign these domains to the corresponding segments of the GroEL sequence. Upon chemical modification of GroEL with N-ethylmaleimide, protease treatment in the presence of ATP or ADP generates GroEL fragments of 15 kDa (N15; residues 1-141) and 40 kDa (C40; residues 153-531). As visualized by scanning transmission electron microscopy, affinity-purified antibodies directed against C40 recognize the outer layers, whereas antibodies against N15 interact with the equatorial portions of the GroEL double-ring. Thus, the two major domains of the subunits in the chaperonin complex are arranged in the order C40-N15:N15-C40. The single-ring chaperonin co-factor GroES interacts with the C40 domain while the ATP-binding site of GroEL is probably close to the junction between N15 and C40.

Antibodies↗