Prion disease is a fatal neurodegenerative disease with no known treatment or cure. The disease is caused by the conformational change of the prion protein (PrP) which leads to its polymerization and causes neuron death. Discovering molecules that can bind to PrP and stabilize its healthy conformation is a promising step towards developing a treatment and/or cure. In order to identify/validate specific ligands for PrP, biophysical and biochemical techniques are combined. First the protocols for protein crystallization, Isothermal Calorimetry (ITC), and Dynamic Light Scattering (DLS) studies were optimized. PrP crystallization conditions were optimized based on the conditions previously described. Homodimer crystals were routinely obtained within days by using sodium chloride as the precipitant agent and cadmium chloride as the additive. Crystals diffracted up to 1.8 Å at the synchrotron source and used to perform fragment screening. DLS experiments monitored the changes in homogeneity and oligomeric states under different concentrations and in the presence of different ligands. Size measurements indicated structural variability within single protein batches which was further characterized as due to the presence of free thiols. Purification protocol under light reducing conditions demonstrated to stabilize the protein, and possibly avoiding structural reshuffling. Determination of binding affinities and complete thermodynamic characterization by ITC were performed in presence of different ligands.

Crystallographic studies of PrP: Discovering potential leads for drug development

255th National Meeting (2018)

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