Johannes Buchner

Publications of Johannes Buchner

• Mechanistic aspects of the Hsp90 phospho-regulation.

  Authors: Joanna Soroka, Johannes Buchner

  Cell cycle (Georgetown, Tex.). 05/2012; 11(10).

  Comment on: Soroka J, et al. Mol Cell 2012; 45:517-28.

• Global Analysis of Phosphoproteome Regulation by the Ser/Thr Phosphatase Ppt1 in Saccharomyces cerevisiae.

  Authors: Thiemo B Schreiber, Nina Mäusbacher, Joanna Soroka, Sebastian K Wandinger, Johannes Buchner, Henrik Daub

  Journal of proteome research. 02/2012;

  Even though protein phosphatases are key regulators of signal transduction, their cellular mechanisms of action are poorly understood. Here, we undertook a large-scale proteomics survey to identifyEven though protein phosphatases are key regulators of signal transduction, their cellular mechanisms of action are poorly understood. Here, we undertook a large-scale proteomics survey to identify cellular protein targets of a serine/threonine phosphatase. We used SILAC-based quantitative MS to measure differences in protein expression and phosphorylation upon ablation of the serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae. Phosphopeptide fractionation by strong cation exchange chromatography combined with immobilized metal affinity chromatography (IMAC) enrichment enabled quantification of more than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient yeast cells. We further quantified the relative expression of 1897 yeast proteins and detected no major protein changes accompanying Ppt1 deficiency. Notably, we found 33 phosphorylation sites to be significantly and reproducibly up-regulated while no phosphorylation events were repressed in cells lacking Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and site-specific fashion. Several of the regulated phosphoproteins were involved in the response to heat stress in agreement with known Ppt1 functions. Additionally, biosynthetic enzymes were particularly prominent among Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the control of various metabolic functions. These results demonstrate the utility of large-scale and quantitative phosphoproteomics to identify cellular sites of serine/threonine phosphatase action in an unbiased manner.

• Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation.

  Authors: Joanna Soroka, Sebastian K Wandinger, Nina Mäusbacher, Thiemo Schreiber, Klaus Richter, Henrik Daub, Johannes Buchner

  Molecular cell. 02/2012; 45(4):517-28.

  Hsp90 is an essential molecular chaperone in the eukaryotic cytosol. Its function is modulated by cochaperones and posttranslational modifications. Importantly, the phosphatase Ppt1 is a dedicatedHsp90 is an essential molecular chaperone in the eukaryotic cytosol. Its function is modulated by cochaperones and posttranslational modifications. Importantly, the phosphatase Ppt1 is a dedicated regulator of the Hsp90 chaperone system. Little is known about Ppt1-dependent phosphorylation sites and how these affect Hsp90 activity. Here, we identified the major phosphorylation sites of yeast Hsp90 in its middle or the C-terminal domain and determined the subset regulated by Ppt1. In general, phosphorylation decelerates the Hsp90 machinery, reduces chaperone function in vivo, sensitizes yeast cells to Hsp90 inhibition and affects DNA repair processes. Modification of one particular site (S485) is lethal, whereas others modulate Hsp90 activity via distinct mechanisms affecting the ATPase activity, cochaperone binding and manipulating conformational transitions in Hsp90. Our mechanistic analysis reveals that phosphorylation of Hsp90 permits a regulation of the conformational cycle at distinct steps by targeting switch points for the communication of remote regions within Hsp90.

• The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop.

  Authors: Andreas B Schmid, Stephan Lagleder, Melissa Ann Gräwert, Alina Röhl, Franz Hagn, Sebastian K Wandinger, Marc B Cox, Oliver Demmer, Klaus Richter, Michael Groll, Horst Kessler, Johannes Buchner

  The EMBO journal. 01/2012;

  Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPRSti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.

• Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach.

  Authors: Nathalie Braun, Martin Zacharias, Jirka Peschek, Andreas Kastenmüller, Juan Zou, Marianne Hanzlik, Martin Haslbeck, Juri Rappsilber, Johannes Buchner, Sevil Weinkauf

  Proceedings of the National Academy of Sciences of the United States of America. 12/2011; 108(51):20491-6.

  The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, itThe molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how "heterogeneity" is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.

• The Hsp90 chaperone machinery: Conformational dynamics and regulation by co-chaperones.

  Authors: Jing Li, Joanna Soroka, Johannes Buchner

  Biochimica et biophysica acta. 09/2011; 1823(3):624-35.

  Hsp90 is a dimeric molecular chaperone required for the activation and stabilization of numerous client proteins many of which are involved in essential cellular processes like signal transductionHsp90 is a dimeric molecular chaperone required for the activation and stabilization of numerous client proteins many of which are involved in essential cellular processes like signal transduction pathways. This activation process is regulated by ATP-induced large conformational changes, co-chaperones and posttranslational modifications. For some co-chaperones, a detailed picture on their structures and functions exists, for others their contributions to the Hsp90 system is still unclear. Recent progress on the conformational dynamics of Hsp90 and how co-chaperones affect the Hsp90 chaperone cycle significantly increased our understanding of the gearings of this complex molecular machinery. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).

• Structural analysis of the interaction between Hsp90 and the tumor suppressor protein p53.

  Authors: Franz Hagn, Stephan Lagleder, Marco Retzlaff, Julia Rohrberg, Oliver Demmer, Klaus Richter, Johannes Buchner, Horst Kessler

  Nature structural & molecular biology. 09/2011; 18(10):1086-93.

  In eukaryotes, the essential dimeric molecular chaperone Hsp90 is required for the activation and maturation of specific substrates such as steroid hormone receptors, tyrosine kinases andIn eukaryotes, the essential dimeric molecular chaperone Hsp90 is required for the activation and maturation of specific substrates such as steroid hormone receptors, tyrosine kinases and transcription factors. Hsp90 is involved in the establishment of cancer and has become an attractive target for drug design. Here we present a structural characterization of the complex between Hsp90 and the tumor suppressor p53, a key mediator of apoptosis whose structural integrity is crucial for cell-cycle control. Using biophysical methods, we show that the human p53 DNA-binding domain interacts with multiple domains of yeast Hsp90. p53 binds to the Hsp90 C-terminal domain in its native-like state in a charge-dependent manner, but it also associates weakly with binding sites in the middle and the N-terminal domains. The fine-tuned interplay between several Hsp90 domains provides the interactions required for efficient chaperoning of p53.

• Closing in on the Hsp90 chaperone-client relationship.

  Authors: Klaus Richter, Johannes Buchner

  Structure (London, England : 1993). 04/2011; 19(4):445-6.

  The molecular chaperone Hsp90 regulates the activity and stability of a set of client proteins. Despite progress in understanding its mechanism, the interaction of Hsp90 with clients has remainedThe molecular chaperone Hsp90 regulates the activity and stability of a set of client proteins. Despite progress in understanding its mechanism, the interaction of Hsp90 with clients has remained enigmatic. Now, in a recent issue of Molecular Cell, Street and coworkers present results that integrate the client in the Hsp90 chaperone cycle.

• Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions.

  Authors: Moritz Marcinowski, Matthias Höller, Matthias J Feige, Danae Baerend, Don C Lamb, Johannes Buchner

  Nature structural & molecular biology. 02/2011; 18(2):150-8.

  The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain-binding protein), togetherThe endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain-binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered C(H)1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1

  Authors: Bjoern O. Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F. Stange, Jan Wehkamp

  Nature. 01/2011; 469:419-423.

  Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal,Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of −200 mV to –300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

• Mixed Hsp90-cochaperone complexes are important for the progression of the reaction cycle.

  Authors: Jing Li, Klaus Richter, Johannes Buchner

  Nature structural & molecular biology. 01/2011; 18(1):61-6.

  The chaperone cycle of heat shock protein-90 (Hsp90) involves progression through defined complexes with different cochaperones. It is still enigmatic how the exchange of cochaperones is regulated.The chaperone cycle of heat shock protein-90 (Hsp90) involves progression through defined complexes with different cochaperones. It is still enigmatic how the exchange of cochaperones is regulated. The first cochaperone entering the cycle is the Hsp90 ATPase inhibitor Sti1 (Hop in human), which later is replaced by a prolyl isomerase (PPIase) and p23. We found, unexpectedly, that one Sti1 molecule is sufficient to completely inhibit the ATPase of the Hsp90 dimer. Upon addition of a PPIase cochaperone to the Hsp90-Sti1 complex, an asymmetric ternary complex is preferentially formed. This PPIase-Hsp90-Sti1 intermediate is important for the progression of the cycle. To expel the bound Sti1, the concerted action of ATP and p23 is required. This mechanism, which is strictly conserved between the yeast and human Hsp90 systems, presents an example of how, in a cyclic process, directionality of assembly and disassembly of protein complexes can be achieved.

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1

  Authors: Bjoern O. Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F. Stange, Jan Wehkamp

  Keystone Symposium, Mucosal Biology: A Fine Balance between Tolerance and Immunity, Vancouver, British Columbia; 01/2011

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1.

  Authors: Bjoern O Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F Stange, Jan Wehkamp

  Nature. 01/2011; 469(7330):419-23.

  Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal,Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200 mV to -300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

• Experimental optimization of protein refolding with a genetic algorithm.

  Authors: Bernd Anselment, Danae Baerend, Elisabeth Mey, Johannes Buchner, Dirk Weuster-Botz, Martin Haslbeck

  Protein science : a publication of the Protein Society. 11/2010; 19(11):2085-95.

  Refolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major bottleneck. As in vitro refolding isRefolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major bottleneck. As in vitro refolding is a complex reaction with a variety of critical parameters, suitable refolding conditions are typically derived empirically in extensive screening experiments. Here, we introduce a new strategy that combines screening and optimization of refolding yields with a genetic algorithm (GA). The experimental setup was designed to achieve a robust and universal method that should allow optimizing the folding of a variety of proteins with the same routine procedure guided by the GA. In the screen, we incorporated a large number of common refolding additives and conditions. Using this design, the refolding of four structurally and functionally different model proteins was optimized experimentally, achieving 74-100% refolding yield for all of them. Interestingly, our results show that this new strategy provides optimum conditions not only for refolding but also for the activity of the native enzyme. It is designed to be generally applicable and seems to be eligible for all enzymes.

• The heat shock response: life on the verge of death.

  Authors: Klaus Richter, Martin Haslbeck, Johannes Buchner

  Molecular cell. 10/2010; 40(2):253-66.

  Organisms must survive a variety of stressful conditions, including sudden temperature increases that damage important cellular structures and interfere with essential functions. In response to heatOrganisms must survive a variety of stressful conditions, including sudden temperature increases that damage important cellular structures and interfere with essential functions. In response to heat stress, cells activate an ancient signaling pathway leading to the transient expression of heat shock or heat stress proteins (Hsps). Hsps exhibit sophisticated protection mechanisms, and the most conserved Hsps are molecular chaperones that prevent the formation of nonspecific protein aggregates and assist proteins in the acquisition of their native structures. In this Review, we summarize the concepts of the protective Hsp network.

• Independent evolution of the core domain and its flanking sequences in small heat shock proteins.

  Authors: Thomas Kriehuber, Thomas Rattei, Thomas Weinmaier, Alexander Bepperling, Martin Haslbeck, Johannes Buchner

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 10/2010; 24(10):3633-42.

  Small heat shock proteins (sHsps) are molecular chaperones involved in maintaining protein homeostasis; they have also been implicated in protein folding diseases and in cancer. In this proteinSmall heat shock proteins (sHsps) are molecular chaperones involved in maintaining protein homeostasis; they have also been implicated in protein folding diseases and in cancer. In this protein family, a conserved core domain, the so-called α-crystallin or Hsp20 domain, is flanked by highly variable, nonconserved sequences that are essential for chaperone function. Analysis of 8714 sHsps revealed a broad variation of primary sequences within the superfamily as well as phyla-dependent differences. Significant variations were found in the number of sHsps per genome, their amino acid composition, and the length distribution of the different sequence parts. Reconstruction of the evolutionary tree for the sHsp superfamily shows that the flanking regions fall into several subgroups, indicating that they were remodeled several times in parallel but independent of the evolution of the α-crystallin domain. The evolutionary history of sHsps is thus set apart from that of other protein families in that two exon boundary-independent strategies are combined: the evolution of the conserved α-crystallin domain and the independent evolution of the N- and C-terminal sequences. This scenario allows for increased variability in specific small parts of the protein and thus promotes functional and structural differentiation of sHsps, which is not reflected in the general evolutionary tree of species.

• Hsp12 is an intrinsically unstructured stress protein that folds upon membrane association and modulates membrane function.

  Authors: Sylvia Welker, Birgit Rudolph, Elke Frenzel, Franz Hagn, Gerhard Liebisch, Gerd Schmitz, Johannes Scheuring, Andreas Kerth, Alfred Blume, Sevil Weinkauf, Martin Haslbeck, Horst Kessler, Johannes Buchner

  Molecular cell. 08/2010; 39(4):507-20.

  Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, includingHsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure. The presence of Hsp12 does not alter the overall lipid composition of the plasma membrane but increases membrane stability.

• Response to Corcos: exceptions to the rules.

  Authors: Matthias J Feige, Linda M Hendershot, Johannes Buchner

  Trends in biochemical sciences. 08/2010;

• The Hsp90 co-chaperone p23 of Toxoplasma gondii: Identification, functional analysis and dynamic interactome determination.

  Authors: Pablo C Echeverria, Maria J Figueras, Malvina Vogler, Thomas Kriehuber, Natalia de Miguel, Bin Deng, Maria C Dalmasso, Dwight E Matthews, Mariana Matrajt, Martin Haslbeck, Johannes Buchner, Sergio O Angel

  Molecular and biochemical parasitology. 08/2010; 172(2):129-40.

  Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. Recently a link between the T. gondii Hsp90 chaperone machinery and parasiteToxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. Recently a link between the T. gondii Hsp90 chaperone machinery and parasite development was observed. Here, the T. gondii Hsp90 co-chaperones p23 and Hip were identified mining the Toxoplasma- database (www.toxodb.org). Their identity was confirmed by domain structure and blast analysis. Additionally, analysis of the secondary structure and studies on the chaperone function of the purified protein verified the p23 identity. Studies of co-immunoprecipitation (co-IP) identified two different types of complexes, one comprising at least Hip-Hsp70-Hsp90 and another containing at least p23-Hsp90. Indirect immunofluorescence assays showed that Hip is localized in the cytoplasm in tachyzoites and as well in bradyzoites. For p23 in contrast, a solely cytoplasmic localization was only observed in the tachyzoite stage whereas nuclear and cytosolic distribution and co-localization with Hsp90 was observed in bradyzoites. These results indicate that the T. gondii Hsp90-heterocomplex cycle is similar to the one proposed for higher eukaryotes, further highlighting the implication of the Hsp90/p23 in parasite development. Furthermore, co-IP experiments of tachyzoite/bradyzoite lysates with anti-p23 antiserum and identification of the complexed proteins together with the use of the curated interaction data available from different source (orthologs and Plasmodium databases) allowed us to construct an interaction network (interactome) covering the dynamics of the Hsp90 chaperone machinery.

• Structural and mechanical hierarchies in the alpha-crystallin domain dimer of the hyperthermophilic small heat shock protein Hsp16.5.

  Authors: Morten Bertz, Jin Chen, Matthias J Feige, Titus M Franzmann, Johannes Buchner, Matthias Rief

  Journal of molecular biology. 07/2010; 400(5):1046-56.

  In biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form sphericalIn biological systems, proteins rarely act as isolated monomers. Association to dimers or higher oligomers is a commonly observed phenomenon. As an example, small heat shock proteins form spherical homo-oligomers of mostly 24 subunits, with the dimeric alpha-crystallin domain as the basic structural unit. The structural hierarchy of this complex is key to its function as a molecular chaperone. In this article, we analyze the folding and association of the basic building block, the alpha-crystallin domain dimer, from the hyperthermophilic archaeon Methanocaldococcus jannaschii Hsp16.5 in detail. Equilibrium denaturation experiments reveal that the alpha-crystallin domain dimer is highly stable against chemical denaturation. In these experiments, protein dissociation and unfolding appear to follow an "all-or-none" mechanism with no intermediate monomeric species populated. When the mechanical stability was determined by single-molecule force spectroscopy, we found that the alpha-crystallin domain dimer resists high forces when pulled at its termini. In contrast to bulk denaturation, stable monomeric unfolding intermediates could be directly observed in the mechanical unfolding traces after the alpha-crystallin domain dimer had been dissociated by force. Our results imply that for this hyperthermophilic member of the small heat shock protein family, assembly of the spherical 24mer starts from folded monomers, which readily associate to the dimeric structure required for assembly of the higher oligomer.