Over the past year, the frequency of Chronic Wasting Disease (CWD) has significantly increased, especially in the small, semi-rural town of Greenville, MI. CWD is an example of a Transmissible Spongiform Encephalopathy (TSE), a disease that is caused by the misfolding of prion proteins that are found in concentrated levels in the central nervous system of biological systems. Little is known about the onset of TSEs besides the fact that a primarily a-helical prion protein forms highly denaturant-resistant b-sheet complexes, which then causes a cascade of misfolding among other prion proteins located near the trigger protein. TSEs do not have a cure and are fatal. This research project was designed to analyze elements of secondary structure in prions that may lead to misfolding. Instead of using prions themselves, a safer alternative – the Shadoo protein – was chosen for analysis. The structure of Shadoo has been shown to be similar to that of prion proteins. Shadoo is also found in biological systems and is hypothesized to assist with the misfolding of prions. The Shadoo plasmid was transformed into competent cells using standard techniques. The protein was expressed successfully as shown by SDS-PAGE and purification of the protein is ongoing. Future goals include analysis of protein folding and stability under native conditions using NMR. Following this, the unfolding of the protein will be observed by using the temperature-variant capabilities of the NMR. The ultimate goal of this experiment is to analyze the differences in Shadoo’s structure as it undergoes unfolding and then refolding to attempt to find a portion of the sequence where the misfolding may originate. This information may be useful to address fatal prion protein activity.

Analysis of the folding and misfolding of Shadoo by nuclear magnetic resonance (NMR)

2019 Spring National Meeting

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