Year: 2013
Type: dissertation
Abstract: Prions are derived from the conversion of a normal, alpha-helical, cellular prion protein (PrPC), to an infectious, beta-sheet rich conformer (PrPSc). This proposition was referred as the protein-only hypothesis where the single protein is responsible for a wide diversity of structures with different pathogenic properties. For decades this hypothesis has motivated the scientific community to decipher the elusive features of this new pathogen. However, many unresolved issues still surround the process of PrP conversion, and the molecular structure of PrPSc is still missing. For structural elucidation of prions by NMR-Spectroscopy, the prerequisite is to obtain extensive amounts of isotope labeled recombinant prions. For this, a novel method had been established in our group called “Selective Shearing Amplification” (SSA), where uniform shear forces were used to optimally balance prion growth and prion fragmentation. It was the key objective of this work to establish SSA for the conformational selection and homogeneous amplification of prion conformations using hamster brain derived prion strains (263K and Sc237) as a seed and native recombinant prion protein as a substrate. A total of 800 mg of unlabelled and isotopically labelled (15N and 13C/15N) recombinant hamster Sc237 and 263K PrP aggregates were produced by SSA. These products were further characterized by SSA, negative stain electron microscopy, mass spectrometry and solid state NMR spectroscopy (ssNMR). Using ssNMR, we characterized conformationally selected rec. PrPres and identified three distinct prion conformations in Sc237, but only one conformation in 263K. Notably the 263K conformation is also present in the Sc237 prion conformational mixture. This indicated that overall seed conformations are not altered by shearing mechanisms, but rather conformation can be selected even from mixed aggregates. Thus, from this work it is evident that a structure determination of prions will be possible.
Author: Gupta, Vandana
Institution: Helmholtz Centre for Infection Research
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2802035613