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

摘要

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 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 probe 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 [1,3-¹³C] 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 yield an estimated strand spacing that is within ~10% of the ~4.7–4.8 Å distances typical for parallel β-sheets.