Cell Stress and Chaperones

, Volume 22, Issue 4, pp 503–515 | Cite as

Chaperone activity of human small heat shock protein-GST fusion proteins

  • Hannah Arbach
  • Caley Butler
  • Kathryn A. McMenimen


Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.


Chaperone Fusion protein Small heat shock protein (sHSP) Protein aggregation Protein-protein interaction Light scattering assay Glutathione-S-transferase (GST) 



We thank Caroline Weber Kinn for initial cloning work and Alina Smithe for assistance with the reading of the manuscript and running assays. We thank the Clare Boothe Luce Foundation for supporting this work and for providing summer support for H.E.A and C.S.B. The National Institutes of Health (NIH) R15 GM120654-01 provided funding for this investigation to K.A.M.

Supplementary material

12192_2017_764_MOESM1_ESM.docx (2 mb)
ESM 1 (DOCX 2090 kb)


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Copyright information

© Cell Stress Society International 2017

Authors and Affiliations

  • Hannah Arbach
    • 1
  • Caley Butler
    • 1
  • Kathryn A. McMenimen
    • 1
  1. 1.Department of ChemistryMount Holyoke CollegeSouth HadleyUSA

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