Exploring small heat shock protein chaperones by crosslinking mass spectrometry

Research output: ThesisDoctoral Thesis (compilation)


Together with other molecular chaperones, small heat shock proteins are key components of the protein quality control system, which is comprised of several hundred proteins and acts to maintain proteome homeostasis in the cell. Small heat shock proteins bind unfolding proteins at an early stage, to prevent these from further unfolding and aggregating. Partially unfolded proteins are being held in a refolding competent state, to be refolded by other chaperones or degraded by the degradation machinery. In the stress response, small heat shock proteins are among the most highly upregulated, preparing the cell to absorb large quantities of partially unfolded proteins. In this way, they form the first line of defence against the threat of protein aggregation under stress conditions. The polydispersity and dynamics of the large small heat shock protein oligomers have complicated their structural and functional characterization. In particular, the molecular mechanism of substrate protein protection remains poorly understood.
The work described in this thesis aims to characterize the molecular interactions between the plant small heat shock protein Hsp21 and model substrate proteins by crosslinking mass spectrometry. The model substrate proteins citrate synthase and malate dehydrogenase, both especially vulnerable to temperature-induced aggregation, were protected from aggregation by Hsp21 and therefore used to investigate the Hsp21-substrate interactions that confer protection. To be able to study the transient Hsp21-substrate interaction by crosslinking mass spectrometry, a workflow was developed based on isotope-labelled lysine-specific crosslinking, nano-LC MALDI-TOF/TOF mass spectrometry, and data analysis with the specialized software FINDX. During the development of this workflow, interactions within Hsp21 itself were characterized as a way to evaluate the method and to learn more about the conformation of Hsp21 in absence of substrate. The interpretation of the identified Hsp21-Hsp21 crosslinks required structural information on the Hsp21 oligomer, which was obtained by single particle negative stain electron microscopy. The combination of these data with native mass spectrometry and homology modelling, led to a structure model of the Hsp21 dodecamer. The in-depth analysis of Hsp21-Hsp21 crosslinks provided a framework for further application of the crosslinking mass spectrometry workflow to the Hsp21-substrate interactions. Finally, Hsp21-substrate crosslinks were identified that support the view that unfolding substrate proteins interact with the intrinsically disordered N-terminal region of the small heat shock protein Hsp21.


  • Wietske Lambert
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biological Sciences


  • cross-linking, mass spectrometry, protein-protein interactions, Hsp21, BS3, DTSSP, MALDI-TOF/TOF, client protein, substrate, aggregation, unfolded protein, chaperone, Small heat shock protein, crosslinking
Original languageEnglish
Awarding Institution
Supervisors/Assistant supervisor
Award date2012 Jun 1
  • Department of Chemistry, Lund University
Print ISBNs978-91-7422-299-9
Publication statusPublished - 2012
Publication categoryResearch

Bibliographic note

Defence details Date: 2012-06-01 Time: 13:15 Place: sal K:B, Kemicentrum, Getingevägen 60, Lund External reviewer(s) Name: Benesch, Justin L.P. Title: Dr. Affiliation: University of Oxford ---

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Related research output

Wietske Lambert, Philip J B Koeck, Emma Åhrman, Pasi Purhonen, Kimberley Cheng, Dominika Elmlund, Hans Hebert & Cecilia Emanuelsson, 2011, In: Protein Science. 20, 2, p. 291-301

Research output: Contribution to journalArticle

Wietske Lambert, Christopher Söderberg, Gudrun Rutsdottir, Wilbert C. Boelens & Cecilia Emanuelsson, 2011, In: Protein Science. 20, 10, p. 1682-1691

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Emma Åhrman, Wietske Lambert, J. Andrew Aquilina, Carol V. Robinson & Cecilia Emanuelsson, 2007, In: Protein Science. 16, 7, p. 1464-1478

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