Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils revealsinterstrand contact regions between assembled beta-amyloid peptide subunits

The assembly of the beta -amyloid peptide (A beta) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural i nformation about fibrillogenesis has remained elusive due to the highly ins oluble, noncrystalline nature of the assembled peptide. X-ray Fiber diffrac tion, infrared spectroscopy, and solid-state NMR studies performed on fibri ls composed of A beta peptides have led to conflicting models of the interm olecular alignment of beta -strands. We demonstrate here the use of photoaf finity cross-linking to determine high-resolution structural constraints on A monomers within amyloid fibrils. A photoreactive A beta (1-40) ligand wa s synthesized by substituting L-p-benzoylphenylatanine (Bpa) for phenylalan ine at position 4 (A beta (1-40) F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS-PAGE of d issolved fibrils revealed the light-dependent formation of a covalent A bet a dimer. Enzymatic cleavage followed by mass spectrometric analysis demonst rated the presence of a dimer-specific ion at MH+ = 1825.9, the predicted m ass of a fragment composed of the N-terminal A beta (1-5) F4Bpa tryptic pep tide covalently attached to the C-terminal A beta (29-40) tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked di mer led to the localization of the photo-cross-link between the ketone of t he Bpa4 side chain and the delta -methyl group of the Met35 side chain., Th e Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel a lignment of Ap peptides within amyloid fibrils.