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F U Hartl

Publications and source records attributed to F U Hartl.

At least 55 records · Page 3Linked to original sources
PubMed1997

Recombination of protein domains facilitated by co-translational folding in eukaryotes.

The evolution of complex genomes requires that new combinations of pre-existing protein domains successfully fold into modular polypeptides. During eukaryotic translation model two-domain polypeptides fold efficiently by sequential and co-translational folding of their domains. In contrast, folding of the same proteins in Escherichia coli is posttranslational, and leads to intramolecular misfolding of concurrently folding domains. Sequential domain folding in eukaryotes may have been critical in the evolution of modular polypeptides, by increasing the probability that random gene-fusion events resulted in immediately foldable protein structures.

Animals↗
PubMed1997

Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent.

The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction.

Amino Acid Sequence↗
PubMed1997

The effect of macromolecular crowding on chaperonin-mediated protein folding.

The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in Escherichia coli. Recent studies in vitro demonstrated that GroES binding to GroEL causes the displacement of unfolded polypeptide into the central volume of the GroEL cavity for folding in a sequestrated environment. Resulting native protein leaves GroEL upon GroES release, whereas incompletely folded polypeptide can be recaptured for structural rearrangement followed by another folding trial. Additionally, each cycle of GroES binding and dissociation is associated with the release of nonnative polypeptide into the bulk solution. Here we show that this loss of substrate from GroEL is prevented when the folding reaction is carried out in the presence of macromolecular crowding agents, such as Ficoll and dextran, or in a dense cytosolic solution. Thus, the release of nonnative polypeptide is not an essential feature of the productive chaperonin mechanism. Our results argue that conditions of excluded volume, thought to prevail in the bacterial cytosol, increase the capacity of the chaperonin to retain nonnative polypeptide throughout successive reaction cycles. We propose that the leakiness of the chaperonin system under physiological conditions is adjusted such that E. coli proteins are likely to complete folding without partitioning between different GroEL complexes. Polypeptides that are unable to fold on GroEL eventually will be transferred to other chaperones or the degradation machinery.

Chaperonin 10↗
PubMed1997

Chaperone-assisted protein folding.

Molecular chaperones of the Hsp70 and chaperonin families are basic constituents of the cellular machinery that mediates protein folding. Recent functional and structural studies corroborate existing models for the mechanism of these components. Highlights of the past year include the X-ray crystallographic analysis of the peptide-binding domain of the Escherichia coli Hsp70 homolog, DnaK, the direct demonstration of protein folding in the central cavity of the chaperonin GroEL, and the visualization of conformational changes in GroEL during the chaperonin folding cycle.

Chaperonins↗
PubMed1996

Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90.

The role of the abundant stress protein Hsp90 in protecting cells against stress-induced damage is not well understood. The recent discovery that a class of ansamycin antibiotics bind specifically to Hsp90 allowed us to address this problem from a new angle. We find that mammalian Hsp90, in cooperation with Hsp70, p60, and other factors, mediates the ATP-dependent refolding of heat-denatured proteins, such as firefly luciferase. Failure to refold results in proteolysis. The ansamycins inhibit refolding, both in vivo and in a cell extract, by preventing normal dissociation of Hsp90 from luciferase, causing its enhanced degradation. This mechanism also explains the ansamycin-induced proteolysis of several protooncogenic protein kinases, such as Raf-1, which interact with Hsp90. We propose that Hsp90 is part of a quality control system that facilitates protein refolding or degradation during recovery from stress. This function is used by a limited set of signal transduction molecules for their folding and regulation under nonstress conditions. The ansamycins shift the mode of Hsp90 from refolding to degradation, and this effect is probably amplified for specific Hsp90 substrates.

Animals↗
PubMed1996

Mechanism of chaperonin action: GroES binding and release can drive GroEL-mediated protein folding in the absence of ATP hydrolysis.

As a basic principle, assisted protein folding by GroEL has been proposed to involve the disruption of misfolded protein structures through ATP hydrolysis and interaction with the cofactor GroES. Here, we describe chaperonin subreactions that prompt a re-examination of this view. We find that GroEL-bound substrate polypeptide can induce GroES cycling on and off GroEL in the presence of ADP. This mechanism promotes efficient folding of the model protein rhodanese, although at a slower rate than in the presence of ATP. Folding occurs when GroES displaces the bound protein into the sequestered volume of the GroEL cavity. Resulting native protein leaves GroEL upon GroES release. A single-ring variant of GroEL is also fully functional in supporting this reaction cycle. We conclude that neither the energy of ATP hydrolysis nor the allosteric coupling of the two GroEL rings is directly required for GroEL/GroES-mediated protein folding. The minimal mechanism of the reaction is the binding and release of GroES to a polypeptide-containing ring of GroEL, thereby closing and opening the GroEL folding cage. The role of ATP hydrolysis is mainly to induce conformational changes in GroEL that result in GroES cycling at a physiologically relevant rate.

Adenosine Diphosphate↗
PubMed1996

Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40.

The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a approximately 7-fold increase in its steady-state ATPase activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70. The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone. As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70. The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese. Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates. Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol. Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.

Adenosine Triphosphatases↗
PubMed1996

Nuclear magnetic resonance solution structure of the human Hsp40 (HDJ-1) J-domain.

The J-domain is a highly conserved domain found in all members of the DnaJ family of molecular chaperones. The three-dimensional structure of a recombinant, uniformly 15N-labeled 77-residue polypeptide containing the complete J-domain from human Hsp40 (HDJ-1) has been determined by nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 876 upper distance constraints derived from nuclear Overhauser effects (NOE) and 173 dihedral angle constraints, a group of 20 conformers representing the solution structure of the HDJ-1 J-domain was computed with the program DIANA and energy-minimized with the program OPAL. The average of the pairwise root-mean-square deviations of the individual NMR conformers relative to the mean coordinates for the backbone atoms N, C2 and C' of residues 4 to 54 and 4 to to 66 is 0.88 and 0.99 A respectively. The molecular architecture includes four helices composed of residues 5 to 9, 15 to 28, 40 to 54 and 60 to 66. A turn composed of residues 10 to 14 links helices I and II, and a loop composed of residues 29 to 39 containing a highly conserved tripeptide HPD (residues 31 to 33) connects the antiparallel helices II and III. The tertiary fold formed by helix I-turn-helix II-loop-helix III forms a closed structural core; the less defined helix IV stands away from the core of the domain. The side-chains of the tripeptide HPD extend out from the core of the structure in the opposite direction from helix IV. The structure supports the hypothesis that the highly conserved tripeptide could play a key role in the interaction of Hsp40 with the molecular chaperone, Hsp70.

Amino Acid Sequence↗
PubMed1996

Molecular chaperones in cellular protein folding.

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.

Animals↗
PubMed1996

Principles of chaperone-assisted protein folding: differences between in vitro and in vivo mechanisms.

Molecular chaperones in the eukaryotic cytosol were shown to interact differently with chemically denatured proteins and their newly translated counterparts. During refolding from denaturant, actin partitioned freely between 70-kilodalton heat shock protein, the bulk cytosol, and the chaperonin TCP1-ring complex. In contrast, during cell-free translation, the chaperones were recruited to the elongating polypeptide and protected it from exposure to the bulk cytosol during folding. Posttranslational cycling between chaperone-bound and free states was observed with subunits of oligomeric proteins and with aberrant polypeptides; this cycling allowed the subunits to assemble and the aberrant polypeptides to be degraded. Thus, folding, oligomerization, and degradation are linked hierarchically to ensure the correct fate of newly synthesized polypeptides.

Actins↗
PubMed1996

Protein folding in the central cavity of the GroEL-GroES chaperonin complex.

The chaperonin GroEL is able to mediate protein folding in its central cavity. GroEL-bound dihydrofolate reductase assumes its native conformation when the GroES cofactor caps one end of the GroEL cylinder, thereby discharging the unfolded polypeptide into an enclosed cage. Folded dihydrofolate reductase emerges upon ATP-dependent GroES release. Other proteins, such as rhodanese, may leave GroEL after having attained a conformation that is committed to fold. Incompletely folded polypeptide rebinds to GroEL, resulting in structural rearrangement for another folding trial in the chaperonin cavity.

Amino Acid Sequence↗
PubMed1996

A zinc finger-like domain of the molecular chaperone DnaJ is involved in binding to denatured protein substrates.

The Escherichia coli heat-shock protein DnaJ cooperates with the Hsp70 homolog DnaK in protein folding in vitro and in vivo. Little is known about the structural features of DnaJ that mediate its interaction with DnaK and unfolded polypeptide. DnaJ contains at least four blocks of sequence representing potential functional domains which have been conserved throughout evolution. In order to understand the role of each of these regions, we have analyzed DnaJ fragments in reactions corresponding to known functions of the intact protein. Both the N-terminal 70 amino acid 'J-domain' and a 35 amino acid glycine-phenylalanine region following it are required for interactions with DnaK. However, only complete DnaJ can cooperate with DnaK and a third protein, GrpE, in refolding denatured firefly luciferase. As demonstrated by atomic absorption and extended X-ray absorption fine structure spectroscopy (EXAFS), the 90 amino acid cysteine-rich region of DnaJ contains two Zn atoms tetrahedrally coordinated to four cysteine residues, resembling their arrangement in the C4 Zn binding domains of certain DNA binding proteins. Interestingly, binding experiments and cross-linking studies indicate that this Zn finger-like domain is required for the DnaJ molecular chaperone to specifically recognize and bind to proteins in their denatured state.

Absorptiometry, Photon↗
PubMed1996

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.

Humans↗
PubMed1996

Protein folding in the cell: competing models of chaperonin function.

The long-standing view that polypeptide chains newly synthesized inside cells fold spontaneously to their functional conformations in an energy-independent fashion derives from the observation that many pure denatured proteins will refold spontaneously in vitro when the denaturant is removed. This view is being challenged by the alternative proposal that in vivo many chains need to be helped to fold correctly by preexisting proteins acting as molecular chaperones, some of which hydrolyse ATP. The need for molecular chaperones arises because of the high concentrations of transiently interacting protein surfaces inside cells permit the formation of incorrect nonfunctional structures. The best-studied family of molecular chaperones are called the chaperonins, the archetypal examples being the GroEL and GroES proteins of Escherichia coli. The chaperonins increase the yield of correctly refolded polypeptide chains, both by decreasing their propensity to aggregate with one another and by allowing polypeptides kinetically trapped in incorrect conformations to make fresh attempts to refold into the functional conformations. The mechanisms by which the chaperonins achieve these remarkable results are currently under debate. This review surveys competing models for chaperonin action, and emphasizes the importance when evaluating these models of considering the intracellular environment in which the chaperonins have evolved to function.

Chaperonin 10↗
PubMed1995

Hip, a novel cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle.

The Hsc70-interacting protein Hip, a tetratricopeptide repeat protein, participates in the regulation of the eukaryotic 70 kDa heat shock cognate Hsc70. One Hip oligomer binds the ATPase domains of at least two Hsc70 molecules dependent on activation of the Hsc70 ATPase by Hsp40. While hydrolysis remains the rate-limiting step in the ATPase cycle, Hip stabilizes the ADP state of Hsc70 that has a high affinity for substrate protein. Through its own chaperone activity, Hip may contribute to the interaction of Hsc70 with various target proteins. We propose a mechanism for the regulation of eukaryotic Hsc70 that is distinct from that of bacterial Hsp70. The Hsc70/Hsp40/Hip system is apparently independent of a GrpE-like nucleotide exchange factor.

Acute-Phase Proteins↗
PubMed1995

Functional significance of symmetrical versus asymmetrical GroEL-GroES chaperonin complexes.

The Escherichia coli chaperonin GroEL and its regulator GroES are thought to mediate adenosine triphosphate-dependent protein folding as an asymmetrical complex, with substrate protein bound within the GroEL cylinder. In contrast, a symmetrical complex formed between one GroEL and two GroES oligomers, with substrate protein binding to the outer surface of GroEL, was recently proposed to be the functional chaperonin unit. Electron microscopic and biochemical analyses have now shown that unphysiologically high magnesium concentrations and increased pH are required to assemble symmetrical complexes, the formation of which precludes the association of unfolded polypeptide. Thus, the functional significance of GroEL:(GroES)2 particles remains to be demonstrated.

Adenosine Triphosphate↗