Small heat shock proteins (sHsps) are oligomeric proteins expressed by cells in response to high temperatures. It is believed that sHsps are produced as a defensive mechanism against temperature stress and act as molecular chaperones by binding and protecting heat-labile proteins from irreversible aggregation. Binding results in the formation of sHsp/substrate complexes from which substrate can later be refolded by ATP-dependent chaperones. Despite past investigations, many aspects of this model remain poorly defined. Results presented here provide new insight into the mechanism of sHsp action. sHsp chaperone activity and sHsp oligomerization are closely linked. Therefore, an understanding of the oligomeric structure, subunit number, and subunit dynamics is essential to understanding sHsp action. Three sHsps were analyzed for these properties: PsHsp18.1 from pea, TaHsp16.9 from wheat, and SynHsp16.6, from the cyanobacterium Synechocystis. In solution, SynHsp16.6 is a duodecamer, while TaHsp16.9 and PsHsp18.1 are dodecamers. An equilibrium between an oligomeric and suboligomeric state was observed for PsHsp18.1 and SynHsp16.6. Increasing temperatures resulted in the reversible dissociation of the TaHsp16.9 oligomer into a suboligomeric species. These results indicate that subunit dynamics are important for sHsp function. Interactions between sHsp and substrate in sHsp/substrate complexes and the mechanism by which substrate is transferred to refolding chaperones are poorly defined. C-terminal affinity-tagged sHsps were used to investigate these issues. This analysis revealed that while some sHsp subunits within sHsp/substrate complexes remain dynamic, complex size remains unchanged and association of substrate with sHsp is not similarly dynamic. These data suggest a model in which ATP-dependent chaperones associate directly with sHsp-bound substrate to initiate refolding. The homologous TaHsp16.9 and PsHsp18.1 are structurally similar. However, TaHsp16.9 interacts differently with substrate and is less effective at protecting substrate than PsHsp18.1. Studies with chimeric sHsps made between PsHsp18.1 and TaHsp16.9 revealed that the N-terminal arm is involved in subunit affinity, substrate protection, and substrate refolding, but interactions between the N-terminal arm and C-terminal domain are also critical for these aspects of chaperone activity. Additionally, the first ten residues of the N-terminal arm play a role in sHsp subunit affinity and substrate protection, but are unimportant for substrate protection.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/280410 |
| Date | January 2003 |
| Creators | Friedrich, Kenneth Lane |
| Contributors | Vierling, Elizabeth |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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