Photoaffinity Cross-Linking of Alzheimer's Disease Amyloid Fibrils Reveals Interstrand Contact Regions between Assembled β-Amyloid Peptide Subunits

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Abstract
The assembly of the β-amyloid peptide (Aβ) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural information about fibrillogenesis has remained elusive due to the highly insoluble, noncrystalline nature of the assembled peptide. X-ray fiber diffraction, infrared spectroscopy, and solid-state NMR studies performed on fibrils composed of Aβ peptides have led to conflicting models of the intermolecular alignment of β-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Aβ monomers within amyloid fibrils. A photoreactive Aβ1-40 ligand was synthesized by substituting l-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Aβ1-40 F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS−PAGE of dissolved fibrils revealed the light-dependent formation of a covalent Aβ dimer. Enzymatic cleavage followed by mass spectrometric analysis demonstrated the presence of a dimer-specific ion at MH+ = 1825.9, the predicted mass of a fragment composed of the N-terminal Aβ1-5 F4Bpa tryptic peptide covalently attached to the C-terminal Aβ29-40 tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked dimer led to the localization of the photo-cross-link between the ketone of the Bpa4 side chain and the δ-methyl group of the Met35 side chain. The Bpa4−Met35 intermolecular cross-link is consistent with an antiparallel alignment of Aβ peptides within amyloid fibrils.