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Johannes Buchner

Publications and source records attributed to Johannes Buchner.

16 recordsLinked to original sources
PubMed2003

NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP, AMP-PNP, geldanamycin, and radicicol.

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.

Adenylyl Imidodiphosphate↗
PubMed2003

Dissection of the contribution of individual domains to the ATPase mechanism of Hsp90.

Hsp90 is a dimeric, ATP-regulated molecular chaperone. Its ATPase cycle involves the N-terminal ATP binding domain (amino acids (aa) 1-272) and, in addition, to some extent the middle domain (aa 273-528) and the C-terminal dimerization domain (aa 529-709). To analyze the contribution of the different domains and the oligomeric state on the progression of the ATPase cycle of yeast Hsp90, we created deletion constructs lacking either the C-terminal or both the C-terminal and the middle domain. To test the effect of dimerization on the ATPase activity of the different constructs, we introduced a Cys residue at the C-terminal ends of the constructs, which allowed covalent dimerization. We show that all monomeric constructs tested exhibit reduced ATPase activity and a decreased affinity for ATP in comparison with wild type Hsp90. The covalently linked dimers lacking only the C-terminal domain hydrolyze ATP as efficiently as the wild type protein. Furthermore, this construct is able to trap the ATP molecule similar to the full-length protein. This demonstrates that in the ATPase cycle, the C-terminal domain can be replaced by a cystine bridge. In contrast, the ATPase activity of the artificially linked N-terminal domains remains very low and bound ATP is not trapped. Taken together, we show that both the dimerization of the N-terminal domains and the association of the N-terminal with the middle domain are important for the efficiency of the ATPase cycle. These reactions are synergistic and require Hsp90 to be in the dimeric state.

Adenosine Triphosphatases↗
PubMed2003

Modulation of the ATPase cycle of BiP by peptides and proteins.

BiP, the Hsp70 homologue of the endoplasmic reticulum, interacts with its non-native substrate proteins in an ATP-dependent manner. This interaction is coupled to the ATPase cycle of the chaperone. Binding of short, synthetic peptides stimulate the ATPase activity of BiP. In previous work, we showed that a stably unfolded antibody domain forms a binary complex with BiP. In this study we made use of this complex to analyse the effect of substrate proteins on the ATPase cycle of BiP. Kinetic constants of the partial reactions of the ATPase cycle were determined without substrate, in the presence of a short binding peptide and in the presence of the antibody domain. We show that, in contrast to smaller peptides, the non-native protein domain decelerates the rate limiting hydrolysis step of the ATPase cycle.

Adenosine Triphosphatases↗
PubMed2003

Sti1 is a novel activator of the Ssa proteins.

The molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop. In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction. With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins. Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro. The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90. Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system. Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1. Thus, Sti1 is a potent novel effector for the Hsp70 ATPase.

Adenosine Triphosphatases↗
PubMed2003

Recombinant expression and purification of Ssa1p (Hsp70) from Saccharomyces cerevisiae using Pichia pastoris.

Heat shock proteins with a molecular mass of 70000 (Hsp70s) are a ubiquitous class of ATP-dependent molecular chaperones involved in the folding of cellular proteins. Sequencing the entire genome of Saccharomyces cerevisiae revealed 14 different genes for Hsp70 proteins in different cellular compartments. Among these 14 Hsp70s, the subclass of Ssa (Ssa1p-Ssa4p) is abundant and essential in the cytosol. Since high yield expression of cytoplasmic Ssa1p is inefficient in Saccharomyces cerevisiae and recombinant expression in E. coli yields low protein levels, we chose Pichia pastoris as the recombinant expression system. In Pichia pastoris, expression levels of Ssa1p are high and Ssa1p is soluble and correctly folded. Also, we present a new protocol for purification of Ssa1p. Previously described purifications include ATP-agarose chromatography leading to Ssa1p partially complexed with ATP. Our optimized purification protocol follows the CiPP strategy (capture, intermediate purification, polishing) avoiding ATP-agarose chromatography, which allows detailed studies on the ATP-dependent Hsp70 functions. We obtained Ssa1p in high purity and 400 times higher quantity compared to previous studies.

Chromatography, Ion Exchange↗
PubMed2003

Analysis of the interaction of small heat shock proteins with unfolding proteins.

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.

Animals↗
PubMed2003

Sti1 is a non-competitive inhibitor of the Hsp90 ATPase. Binding prevents the N-terminal dimerization reaction during the atpase cycle.

The molecular chaperone Hsp90 is known to be involved in the activation of key regulatory proteins such as kinases, steroid hormone receptors, and transcription factors in an ATP-dependent manner. During the chaperone cycle, Hsp90 has been found associated with the partner protein Hop/Sti1, which seems to be required for the progression of the cycle. However, little is known about its specific function. Here we have investigated the interaction of Sti1 from Saccharomyces cerevisiae with Hsp90 and its influence on the ATPase activity. We show that the inhibitory mechanism of Sti1 on the ATPase activity of Hsp90 is non-competitive. Sti1 binds to the N- and C-terminal part of Hsp90 and prevents the N-terminal dimerization reaction that is required for efficient ATP hydrolysis. The first 24 amino acids of Hsp90, a region shown previously to be important for the association of the N-terminal domains and stimulation of ATP hydrolysis, seems to be important for this interaction.

Adenosine Triphosphatases↗
PubMed2002

p53 contains large unstructured regions in its native state.

The human tumor suppressor protein p53 is understood only to some extent on a structural level. We performed a comprehensive biochemical and biophysical structure-function analysis of p53 full-length protein and p53 fragments. The analysis showed that p53 and the fragments investigated form stable functional units. Full-length p53 and the tetrameric fragment N93p53 (residues 93-393) are, however, destabilized significantly compared to the monomeric core domain (residues 94-312) and the monomeric fragment p53C312 (residues 1-312). At the physiological temperature of 37 degrees C and in the absence of modifications or stabilizing partners, wild-type p53 is more than 50% unfolded correlating with a 75% loss in DNA-binding activity. Furthermore the analysis of CD spectra revealed that full-length p53 contains large unstructured regions in its N and C-terminal parts. Our results indicate that full-length p53 is a modular protein consisting of defined structured and unstructured regions. We propose that p53 belongs to the growing family of loosely folded or partially unstructured native proteins. The lack of a rigid structure combined with the low overall stability may allow the physiological interaction of p53 with a multitude of partner proteins and the regulation of its turnover.

Calorimetry↗
PubMed2002

N-terminal residues regulate the catalytic efficiency of the Hsp90 ATPase cycle.

Hsp90 is an abundant molecular chaperone involved in a variety of cellular processes ranging from signal transduction to viral replication. The function of Hsp90 has been shown to be dependent on its ability to hydrolyze ATP, and in vitro studies suggest that the dimeric nature of Hsp90 is critical for this activity. ATP binding occurs at the N-terminal domains of the Hsp90 dimer, whereas the main dimerization site resides in the very C-terminal domain. ATP hydrolysis is performed in a series of conformational changes. These include the association of the two N-terminal domains, which has been shown to stimulate the hydrolysis reaction. In this study, we set out to identify regions in the N-terminal domain that are important for this interaction. We show that N-terminal deletion variants of Hsp90 are severely impaired in their ability to hydrolyze ATP. However, nucleotide binding of these constructs is similar to that of the wild type protein. Heterodimers of the Hsp90 deletion mutants with wild type protein showed that the first 24 amino acids play a crucial role during the ATPase reaction, because their deletion abolishes the trans-activation between the two N-terminal domains. We propose that the turnover rate of Hsp90 is decisively controlled by intermolecular interactions between the N-terminal domains.

Adenosine Diphosphate↗
PubMed2002

Folding and oxidation of the antibody domain C(H)3.

The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge.

Alkylation↗
PubMed2002

Interaction of the chaperone BiP with an antibody domain: implications for the chaperone cycle.

BiP is an Hsp70 homologue found in the endoplasmic reticulum of eukaryotic cells. Like other Hsp70 chaperones, BiP interacts with its substrate proteins in an ATP-dependent manner. The functional analysis has so far been performed mainly with short, synthetic peptides. Here, we present an experimental system that allows to study the partial reactions of the BiP chaperone cycle for a natural substrate protein domain in its soluble, stably unfolded conformation. This unfolded antibody domain forms a binary complex with BiP in the absence of ATP. The dissociation of the BiP dimer seems to be the rate-limiting step in this reaction. The BiP-C(H)3 complexes dissociate rapidly in the presence of ATP. The affinity for BiP-binding peptides and the non-native antibody domain was determined to be similar, suggesting that only the peptide binding site is involved in these interactions. Furthermore, these results imply that, also in the context of the antibody domain, an extended peptide sequence is recognized. However, the accessibility of the BiP-binding site in the non-native protein seems to influence the kinetics of complex formation.

Adenosine Diphosphate↗
PubMed2002

Intradomain disulfide bonds impede formation of the alternatively folded state of antibody chains.

Antibodies undergo significant conformational changes upon acidification, leading to the formation of an alternatively folded state. Here, we analyzed the conformation of MAK 33 Fab and its light chain at acidic pH, both in the reduced and oxidized form. At acidic pH, the proteins exhibited a highly structured, but non-native conformation, corresponding to the alternatively folded state, previously described for the intact antibody. However, the requirements to form this alternative structure were different for the oxidized and reduced protein. Whereas in the oxidized form of the immunoglobulin light chain the alternatively folded state could only be detected at pH<1.4, the reduced light chain already adopted this structure at pH 2. Thermal denaturation measurements revealed that, surprisingly, the alternatively folded state of the reduced light chain was more stable than that of the oxidized protein at pH 1.4. This indicates that the intradomain disulfide bonds, which stabilize the native state of antibody domains, impede the formation of the alternatively folded state.

Animals↗
PubMed2002

Refolding and structural characterization of the human p53 tumor suppressor protein.

The human tumor suppressor p53 is a conformationally flexible and functionally complex protein that is only partially understood on a structural level. We expressed full-length p53 in the cytosol of Escherichia coli as inclusion bodies. To obtain active, recombinant p53, we varied renaturation conditions using DNA binding activity and oligomeric state as criteria for successful refolding. The optimized renaturation protocol allows the refolding of active, DNA binding p53 with correct quaternary structure and domain contact interfaces. The purified protein could be allosterically activated for DNA binding by addition of a C-terminally binding antibody. Analytical gelfiltration and chemical cross-linking confirmed the tetrameric quaternary structure and the spectroscopic analysis of renatured p53 by fluorescence and circular dichroism, suggested that native p53 is partially unstructured.

Circular Dichroism↗
PubMed2002

Molecular chaperones--cellular machines for protein folding.

Proteins are linear polymers synthesized by ribosomes from activated amino acids. The product of this biosynthetic process is a polypeptide chain, which has to adopt the unique three-dimensional structure required for its function in the cell. In 1972, Christian Anfinsen was awarded the Nobel Prize for Chemistry for showing that this folding process is autonomous in that it does not require any additional factors or input of energy. Based on in vitro experiments with purified proteins, it was suggested that the correct three-dimensional structure can form spontaneously in vivo once the newly synthesized protein leaves the ribosome. Furthermore, proteins were assumed to maintain their native conformation until they were degraded by specific enzymes. In the last decade this view of cellular protein folding has changed considerably. It has become clear that a complicated and sophisticated machinery of proteins exists which assists protein folding and allows the functional state of proteins to be maintained under conditions in which they would normally unfold and aggregate. These proteins are collectively called molecular chaperones, because, like their human counterparts, they prevent unwanted interactions between their immature clients. In this review, we discuss the principal features of this peculiar class of proteins, their structure-function relationships, and the underlying molecular mechanisms.

Chaperonin 10↗
PubMed2002

Wheat FKBP73 functions in vitro as a molecular chaperone independently of its peptidyl prolyl cis-trans isomerase activity.

Peptidyl-prolyl cis-trans isomerases (PPIases) catalyse protein folding by accelerating the slow step of cis-trans isomerisation of peptidyl-prolyl bonds. Wheat (Triticum aestivum L.) FKBP73 (wFKBP73) is a peptidyl-prolyl cis-trans isomerase belonging to the FK506-binding protein (FKBP) family. It comprises three FKBP12-like domains, tetratricopeptide repeats and a calmodulin-binding domain (CaMbd). In vitro studies indicated that wFKBP73 possesses PPIase activity, binds calmodulin and forms a heterocomplex with mammalian p23 and wheat Hsp90 in wheat-germ lysate. To further study the role of wFKBP73 we have analysed its chaperone properties. Using the thermal unfolding and aggregation of citrate synthase (CS) as a model system, we have shown that the plant wFKBP73 exhibits chaperone activity, being able to suppress CS aggregation independently of its PPIase activity. The wFKBP73 interacts transiently with non-native CS and slows down its inactivation kinetics, whereas the mammalian homologue, hFKBP52 binds tightly to CS and does not affect its rate of inactivation. Hence, the first plant FKBP shown to function as a molecular chaperone has a mode of action different from that of the mammalian FKBP52.

Calmodulin-Binding Proteins↗