Intermolecular Alignment in Y145Stop Human Prion Protein Amyloid Fibrils Probed by Solid-State NMR Spectroscopy

The Y145Stop mutant of human prion protein, huPrP23–144, has been linked to PrP cerebral amyloid angiopathy, an inherited amyloid disease, and also serves as a valuable in vitro model for investigating the molecular basis of amyloid strains. Prior studies of huPrP23–144 amyloid by magic-angle-spinning (MAS) solid-state NMR spectroscopy revealed a compact β-rich amyloid core region near the C-terminus and an unstructured N-terminal domain. Here, with the focus on understanding the higher-order architecture of huPrP23–144 fibrils, we probed the intermolecular alignment of β-strands within the amyloid core using MAS NMR techniques and fibrils formed from equimolar mixtures of 15N-labeled protein and 13C-huPrP23–144 prepared with [1,3-13C2] or [2-13C]glycerol. Numerous intermolecular correlations involving backbone atoms observed in 2D 15N–13C spectra unequivocally suggest an overall parallel in-register alignment of the β-sheet core. Additional experiments that report on intermolecular 15N–13CO and 15N–13Cα dipolar couplings yielded an estimated strand spacing that is within ∼10% of the distances of 4.7–4.8 Å typical for parallel β-sheets.