Molecular basis for diversification of yeast prion strain conformation.

Self-propagating ß-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Progress towards structural understanding of infectious sheep PrP-amyloid.

The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct ß-sheet core C-terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the ß-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.