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Concept: Chaperonin


Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that associates dynamically with various co-chaperones during its chaperone cycle. Here we analyzed the role of the activating co-chaperone Aha1 in the progression of the yeast Hsp90 chaperone cycle and identified a critical ternary Hsp90 complex containing the co-chaperones Aha1 and Cpr6. Aha1 accelerates the intrinsically slow conformational transitions of Hsp90 to an N-terminally associated state but does not fully close the nucleotide-binding pocket yet. Cpr6 increases the affinity between Aha1 and Hsp90 and further stimulates the Hsp90 ATPase activity. Synergistically, Aha1 and Cpr6 displace the inhibitory co-chaperone Sti1 from Hsp90. To complete the cycle, Aha1 is released by the co-chaperone p23. Thus, at distinct steps during the Hsp90 chaperone cycle, co-chaperones selectively trap statistically distributed Hsp90 conformers and thus turn Hsp90 into a deterministic machine.

Concepts: Chaperonin, Protein, Proteins, Proteasome, Hsp90, Co-chaperone, Heat shock protein, Chaperone


The mitochondrial Hsp70 (mtHsp70) mediates essential functions for mitochondrial biogenesis like import and folding of proteins. In these processes the chaperone cooperates with co-chaperones, the presequence translocase and other chaperone systems. The chaperonin Hsp60 together with its co-factor Hsp10 catalyzes folding of a subset of mtHsp70 client proteins. Hsp60 forms heptameric ring structures, which provide a cavity for protein folding. How the Hsp60 rings are assembled is poorly understood. In a comprehensive interaction study we found that mtHsp70 associates with Hsp60 and Hsp10. Surprisingly, mtHsp70 interacts with Hsp10 independently of Hsp60. The mtHsp70-Hsp10 complex binds to unassembled Hsp60 precursor to promote its assembly into the mature Hsp60 complexes. We conclude that coupling to Hsp10 recruits mtHsp70 to mediate biogenesis of the heptameric Hsp60 rings.

Concepts: Enzyme, Metabolism, Protein structure, Protein, Chaperonin, Protein folding, Chaperone, Heat shock protein


Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the aggregation of unfolding proteins during proteotoxic stress. In Caenorhabditis elegans, Sip1 is the only sHsp exclusively expressed in oocytes and embryos. Here, we demonstrate that Sip1 is essential for heat shock survival of reproducing adults and embryos. X-ray crystallography and electron microscopy revealed that Sip1 exists in a range of well-defined globular assemblies consisting of two half-spheres, each made of dimeric “spokes.” Strikingly, the oligomeric distribution of Sip1 as well as its chaperone activity depend on pH, with a trend toward smaller species and higher activity at acidic conditions such as present in nematode eggs. The analysis of the interactome shows that Sip1 has a specific substrate spectrum including proteins that are essential for embryo development.

Concepts: Hsp90, DNA, Chaperonin, Co-chaperone, Developmental biology, Chaperone, Protein, Heat shock protein


Noncovalently “stacked” tetramethylrhodamine (TMR) dimers have been used to both report and perturb the allosteric equilibrium in GroEL. A GroEL mutant (K242C) has been labeled with TMR, close to the peptide-binding site in the apical domain, such that TMR molecules on adjacent subunits are able to form dimers in the T allosteric state. Addition of ATP induces the transition to the R state and the separation of the peptide-binding sites, with concomitant unstacking of the TMR dimers. A statistical analysis of the spectra allowed us to compute the number and orientation of TMR dimers per ring as a function of the average number of TMR molecules per ring. The TMR dimers thus serve as quantitative reporter of the allosteric state of the system. The TMR dimers also serve as a surrogate for substrate protein, substituting in a more homogeneous, quantifiable manner for the heterogeneous intersubunit, intraring, noncovalent cross-links provided by the substrate protein. The characteristic stimulation of the ATPase activity by substrate protein is also mimicked by the TMR dimers. Using an expanded version of the nested cooperativity model, we determine values for the free energy of the TT to TR and TR to RR allosteric equilibria to be 27 ± 11 and 46 ± 2 kJ/mol, respectively. The free energy of unstacking of the TMR dimers was estimated at 2.6 ± 1.0 kJ/mol dimer. These results demonstrate that GroEL can perform work during the T to R transition, supporting the iterative annealing model of chaperonin function.

Concepts: Kinesin, Sociology, Molecule, Antibody, Statistics, Chaperonin, Enzyme kinetics, Mathematics


Populations of Streptococcus pneumoniae (SP) are typically structured into groups of closely related organisms or lineages, but it is not clear whether they are maintained by selection or neutral processes. Here, we attempt to address this question by applying a machine learning technique to SP whole genomes. Our results indicate that lineages evolved through immune selection on the groEL chaperone protein. The groEL protein is part of the groESL operon and enables a large range of proteins to fold correctly within the physical environment of the nasopharynx, thereby explaining why lineage structure is so stable within SP despite high levels of genetic transfer. SP is also antigenically diverse, exhibiting a variety of distinct capsular serotypes. Associations exist between lineage and capsular serotype but these can be easily perturbed, such as by vaccination. Overall, our analyses indicate that the evolution of SP can be conceptualized as the rearrangement of modular functional units occurring on several different timescales under different pressures: some patterns have locked in early (such as the epistatic interactions between groESL and a constellation of other genes) and preserve the differentiation of lineages, while others (such as the associations between capsular serotype and lineage) remain in continuous flux.

Concepts: Protein, Chaperone, Virus, Gene expression, DNA, Chaperonin, Heat shock protein, Gene


The isolated apical domain of the E. coli GroEL subunit displays the ability to suppress the irreversible fibrillation of numerous amyloid-forming polypeptides. In previous experiments, we have shown that mutating Gly192 (located at hinge II that connects the apical domain and the intermediate domain) to a tryptophan results in an inactive chaperonin whose apical domain is disoriented. In this study, we have utilized this disruptive effect of Gly192 mutation to our advantage, by substituting this residue with amino acid residues of varying Van der Waals volumes with the intent to modulate the affinity of GroEL toward fibrillogenic peptides. The affinities of GroEL toward fibrillogenic polypeptides such as Aβ1-40 peptide and α-synuclein increased in accordance to the increased Van der Waals volume of the substituent amino acid side chain in the G192X mutants. When we compared the effects of wild-type GroEL and selected GroEL G192X mutants on α-synuclein fibril formation, we found that the effects of the chaperonin on α-synuclein fibrillation were different; the wild-type chaperonin caused changes in both the initial lag phase and the rate of fibril extension, while the effects of the G192X mutants were more specific toward the nucleus-forming lag phase. The chaperonins also displayed differential effects on α-synuclein fibril morphology, suggesting that through mutation of Gly192, we may induce changes to the intermolecular affinities between GroEL and α-synuclein, that lead to more efficient fibril suppression, and in specific cases, modulation of fibril morphology.

Concepts: DNA, Module, Chaperonin, Antioxidant, Bacteria, Peptide, Protein, Amino acid


The aggregation of α-synuclein (α-syn) into amyloid fibrils is associated with neurodegenerative diseases, collectively referred to as the α-synucleinopathies. In vivo, molecular chaperones, such as the small heat-shock proteins (sHsps), normally act to prevent protein aggregation; however, it remains to be determined how aggregation-prone α-syn evades sHsp chaperone action leading to its disease-associated deposition. This work examines the molecular mechanism by which two canonical sHsps, αB-crystallin (αB-c) and Hsp27, interact with aggregation-prone α-syn to prevent its aggregation in vitro. Both sHsps are very effective inhibitors of α-syn aggregation, but no stable complex between the sHsps and α-syn was detected, indicating that the sHsps inhibit α-syn aggregation via transient interactions. Moreover, the ability of these sHsps to prevent α-syn aggregation was dependent on the kinetics of aggregation; the faster the rate of aggregation (shorter the lag phase), the less effective the sHsps were at inhibiting fibril formation of α-syn. Thus, these findings indicate that the rate at which α-syn aggregates in cells may be a significant factor in how it evades sHsp chaperone action in the α-synucleinopathies.

Concepts: Protein folding, Hsp90, Chaperonin, Proteasome, Co-chaperone, Protein, Chaperone, Heat shock protein


In E. coli cells, rescue of non-native proteins and promotion of native state structure is assisted by the chaperonin GroEL. An important key to this activity lies in the structure of the apical domain of GroEL (GroEL-AD) (residue 191-376), which recognizes and binds non-native protein molecules through hydrophobic interactions. In this study, we investigated the effects of GroEL-AD on the aggregation of various client proteins (α-Synuclein, Aβ42, and GroES) that lead to the formation of distinct protein fibrils in vitro. We found that GroEL-AD effectively inhibited the fibril formation of these three proteins when added at concentrations above a critical threshold; the specific ratio differed for each client protein, reflecting the relative affinities. The effect of GroEL-AD in all three cases was to decrease the concentration of aggregate-forming unfolded client protein or its early intermediates in solution, thereby preventing aggregation and fibrillation. Binding affinity assays revealed some differences in the binding mechanisms of GroEL-AD toward each client. Our findings suggest a possible applicability of this minimal functioning derivative of the chaperonins (the “minichaperones”) as protein fibrillation modulators and detectors.

Concepts: Protein folding, GroES, Gene, Affinity, Escherichia coli, GroEL, Chaperonin, Protein


Molecular chaperones, also known as heat-shock proteins, refold misfolded proteins and help other proteins reach their native conformation. Thanks to these abilities, some chaperones, such as the Hsp90 protein or the chaperonin GroEL, can buffer the deleterious phenotypic effects of mutations that alter protein structure and function. Hsp70 chaperones use a chaperoning mechanism different from that of Hsp90 and GroEL, and it is not known whether they can also buffer mutations. Here, we show that they can. To this end, we performed a mutation accumulation experiment in Escherichia coli, followed by whole-genome resequencing. Overexpression of the Hsp70 chaperone DnaK helps cells cope with mutational load and completely avoid the extinctions we observe in lineages evolving without chaperone overproduction. Additionally, our sequence data show that DnaK overexpression increases mutational robustness, the tolerance of its clients to nonsynonymous nucleotide substitutions. We also show that this elevated mutational buffering translates into differences in evolutionary rates on intermediate and long evolutionary time scales. Specifically, we studied the evolutionary rates of DnaK clients using the genomes of E. coli, Salmonella enterica, and 83 other gamma-proteobacteria. We find that clients that interact strongly with DnaK evolve faster than weakly interacting clients. Our results imply that all three major chaperone classes can buffer mutations and affect protein evolution. They illustrate how an individual protein like a chaperone can have a disproportionate effect on the evolution of a proteome.

Concepts: Endoplasmic reticulum, Chaperonin, Escherichia coli, Evolution, Chaperone, Protein folding, Heat shock protein, Protein


Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.

Concepts: Chaperonin, Proteins, Molecular dynamics, Protein targeting, Chaperone, Protein structure, Protein, Protein folding