Cryo‐ <scp>EM</scp> Analysis of Bovine <scp>PrP</scp> (108–144) Fibrils and Implications for Sequence‐Dependent Species Barriers

ABSTRACT The central dogma of protein folding theory is that the amino acid sequence determines protein structure. However, whether sequence variations alter protein misfolding remains poorly understood, largely due to the challenges of studying misfolded products such as amyloid fibrils. Recent advances in cryo‐electron microscopy (cryo‐EM) have enabled structural resolution of infectious prions isolated from mouse, hamster, human, and cervid brains. For bovine prions, which are known to cause cross‐species transmission from cattle to humans, structural characterization remains elusive due to biosafety concerns associated with highly infectious materials. Here, we investigate a non‐infectious bovine prion peptide spanning residues 108–144, a region crucial for fibril formation. Using a synthetic bPrP(108–144) peptide, cryo‐EM revealed intrinsic fibril polymorphism, with two coexisting fibril morphologies. The dominant Type I species consists of two protofilaments, with a core spanning residues 108–144 and two β‐strands located at residues 115–123 and 138–140. A second, less abundant morphology represents an alternative assembly state, indicating multiple conformational basins in the bPrP(108–144) misfolding landscape. Notably, despite differing by only three residues, the bPrP(108–144) Type I structure is distinct from previously characterized hamster prion peptide fibrils, suggesting that minor sequence variations can be structurally amplified in the absence of stabilizing N‐terminal regions. Together, these findings establish the PrP(108–144) fragment as a sensitive structural probe for dissecting sequence‐dependent conformational variability and species transmission barriers in prion misfolding.