Cross-beta structure of an amyloid-forming peptide studied by electron nano-crystallography

The seven residue peptide GNNQQNY from the N-terminal region of the yeast prion protein Sup35, which forms both amyloid fibres and highly ordered microcrystals, provides a model system for characterizing the structure and stability of the elusive cross-beta amyloid conformation. Microcrystals of this peptide, which have largest dimension ~1µm in the cross-beta fibre axis direction, diffract electrons to ultra high resolution (<0.5Å spacing). The sharp 4.86Å layer-line spacing (with no trace of a 9.72Å spacing) establishes that the peptide chains form parallel β-sheets. The space group is P212121 with cell dimensions ranging from |a| = 22.7-21.2, |b| = 39.9-39.3, |c| = 4.89-4.86Å for the wet to dried state. The relative intensities of the Bragg reflections determined from the electron diffraction data indicate that the backbones of the four peptides in the unit cell are aligned closely parallel to the a-axis, with the cross-beta sheets connected in two pairs related by the crystal 2-fold screw axes. Comparison of the cell volume with that calculated for the four peptides implies that water may occupy only 10-15% of the volume in the hydrated state. This nearly anhydrous packing can account for the insolubility of the crystalline aggregates of this hydrophilic peptide, since the activation energy for rehydration may be extremely high. Water excluding packing of paired cross- β peptide segments in thin protofilaments may be a general characteristic of the wide variety of anomalously stable pathological amyloid assemblies.