Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrP C , into a plethora of assemblies, PrP Sc , associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrP Sc structures. However, the degree to which intra- and inter-strain PrP Sc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrP Sc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrP Sc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrP Sc particles. We found that each strain had a continuum of PrP Sc assemblies, with strong correlation between PrP Sc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrP Sc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrP Sc took place at a hydrodynamic radius (R h ) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrP Sc subpopulations and demonstrate that while PrP Sc quaternary structure is a major contributor to PrP Sc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrP Sc particles from maintaining proteinase K resistance below an R h of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.