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Structural features and interactions of substrates complexed with molecular chaperones

Ungelenk, Sophia Maria

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Protein misfolding and aggregation perturbs cellular functions and is involved in aging and numerous medical disorders. In cells, the first line of defense is the association of deleterious aggregating proteins with small Heat shock proteins (sHsp). These oligomeric, ATP-independent chaperones sequester misfolded proteins into complexes and facilitate subsequent substrate solubilization and refolding by ATP-dependent chaperones. The cytosol of S. cerevisiae contains two sHsps: Hsp42 is constitutively active, while Hsp26 is activated at elevated temperatures. In my thesis, I wanted to elucidate how sHsps change the structure of aggregates, facilitating substrate reactivation. To this end, I studied the impact of Hsp26 and Hsp42 incorporation on the architecture of heat-induced aggregates by amide hydrogen exchange (HX). I established the experimental conditions for HX of heat-induced protein aggregates using thermolabile malate dehydrogenase (MDH) as model substrate. My data show that the formation of heat-induced Hsp26/MDH or Hsp42/MDH complexes has profound impact on the MDH structure. In the aggregated state formed in absence of sHsps, almost the entire MDH polypeptide becomes accessible to HX, reflecting global, large misfolding. In contrast, a more protected form of MDH is detected when complexed with Hsp26 or Hsp42. I observed that the mass spectra of many MDH peptides derived from sHsp/MDH complexes exist as a mixture of two populations after HX: a native-like and an aggregate-like population. Higher excess of sHsps promoted the native-like state. Single-molecule experiments confirmed the binding of sHsps to near native substrate folds. Furthermore, FRET experiments showed that sHsps increase the spacing between MDH molecules in sHsp/MDH complexes, preventing intermolecular contacts of misfolded MDH species. Finally, crosslinking approaches identified peripheral, surface-exposed MDH sites showing high HX as major sHsp binding sites. Summarized, these findings indicate that sHsps capture early unfolding intermediates of substrates and keep parts of the protein in a native-like state. This activity of sHsps might facilitate chaperone-dependent disaggregation. I then investigated how the two sHsps of yeast interact with their substrates. The N-terminal extensions (NTE) of both yeast sHsps were found to be the major substrate interaction sites. Compared to all known sHsps, the NTE of Hsp42 is unusually elongated and it was shown to be involved in the organized deposition of misfolded proteins at CytoQ (cytosolic quality control compartment). Hsp42 NTE harbors the two prototypes of intrinsically disordered domains (IDD): a prion-like and an unstructured subdomain. IDDs play important roles in the formation of membrane-free compartments due to their ability to self-associate and to coalesce into inclusions. In this study, the roles of both NTE subdomains in CytoQ formation and Hsp42 chaperone activity were investigated. We found that the prion-like domain of Hsp42 has a dual function: It binds misfolded substrate proteins and triggers CytoQ formation. The unstructured domain is dispensable for CytoQ formation, but it has a regulatory function, controlling Hsp42 localization and CytoQ numbers. Deletion of the unstructured domain increases Hsp42 substrate interaction and holdase activity, i.e. the prevention of tight contacts between misfolded species. Together, the presented data show that the prion-like domain of Hsp42 is essential for CytoQ formation, extending the role of prion-like domains in inclusion formation from RNA granules to protein aggregates and emphasizing their crucial contributions to protein phase transitions. In a second part of my thesis I studied how the Hsp70 chaperone system interacts with RepE, a dimeric replication initiation protein in E. coli. The disassembly of RepE seems mechanistically related to the disaggregation process. As a dimer RepE represses its own transcription, as a monomer it initiates the replication of the mini-F plasmid. Monomerization is mediated by the DnaK chaperone system. So far, it remained elusive, how components of the DnaK chaperone system interact with RepE and how they change its structure, leading to the disassembly of the RepE dimer. In this study the binding of DnaK and DnaJ to dimeric RepE wt and to RepE54, a constitutively monomeric variant, was studied by HX. HX analysis of RepE wt revealed a putative DnaK binding site and conformational changes induced by chaperones. Only dimeric RepE wt, but not monomeric RepE54, interacts with DnaJ. In contrast, both oligomeric states of RepE were able to bind DnaK – at least in absence of DNA. In presence of their respective DNA-binding elements, the binding of DnaK was prevented, most likely due to sterical hindrance as the DNA and the putative DnaK binding sites in RepE are in close proximity. The binding of DnaJ probably occurs in aa 96-116, and it destabilized parts of the DNA binding region in RepE, indicating conformational changes. Although interaction with DnaJ was shown to enhance the binding affinity of RepE to DNA, the DnaJ-induced conformational change might enable DnaK to access its binding site. Crosslinking experiments, however, showed that DnaJ binding is not sufficient to allow for interaction of DnaK with DNA-complexed RepE wt. Only concomitant presence of DnaJ and GrpE enabled DnaK to interact with DNA-bound RepE wt. HX revealed, that concerted binding of DnaJ and DnaK causes substantial conformational changes in RepE: Destabilization of the C-terminal region and stabilization in helix α4 near the dimer interface. The latter might be implicated in the monomerization of RepE wt. In summary, my results provide major contributions to elucidate the chaperone-mediated RepE monomerization process.

Item Type: Dissertation
Supervisor: Bukau, Prof. Dr. Bernd
Date of thesis defense: 7 May 2015
Date Deposited: 01 Jul 2015 09:59
Date: 2015
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
Controlled Keywords: Chaperone, Protein quality control, small heat shock proteins
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