Date of Award
Small heat shock proteins (sHsps) are ubiquitous proteins that act as molecular chaperones under heat stress in a vast range of organisms. These proteins bind and hold in a stable conformation partially unfolded or heat-denatured intracellular proteins. Under non-stressful conditions, sHsps can be found in oligomeric assemblies with the oligomers acting as storage units for dimers and monomers. As temperatures increase to stressful levels, these oligomers dissociate into suboligomeric species, the dimer being the preferred species, to permit binding of substrate. Following substrate binding, oligomeric species may reform, and contain a variable number of sHsp:substrate complexes depending on the host organism, the identity of the substrate, the identity of the sHsp, and other factors. Subunit exchange occurs prior to and after the binding of substrate, and appears to be an important factor in the chaperone mechanism. The rate of subunit exchange as well as the change in rates over time at various temperatures can be quantified using fluorescence resonance energy transfer (FRET). An understanding of the mechanism and efficiency of subunit exchange may increase knowledge of the nature of sHsps, and shed more light on the importance of this seemingly vital component of their chaperone mechanism. Though sHsp 17.0 comes from Zea maize, information from this study will benefit similar research concerning other sHsps. These proteins have been implicated in a number of human diseases including ALS, Parkinson’s disease, and desmin-related myopathy, but the sHsps also show great potential in therapeutic applications. In this study we attempted to isolate and purify sHsp 17.0 to study its chaperone abilities with the model substrate citrate synthase (CS), and to measure subunit exchange using FRET. Protein purification was incomplete, but will be improved in the near future. In addition, progress was made in optimizing CS aggregation assays using a small volume cuvette. The findings of this study will undoubtedly inform the future undertakers of this work.
Schleich, Michele and Tims, Hannah, "Isolation of Class II sHsp 17.0 from Zea maize for Future Studies in Chaperone Activity and Subunit Exchange" (2010). Honors Projects and Presentations: Undergraduate. 110.