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.
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