Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils reveals interstrand contact regions between assembled beta-amyloid peptide subunits.
The assembly of the beta-amyloid peptide (Abeta) 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 Abeta peptides have led to conflicting models of the intermolecular alignment of beta-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Abeta monomers within amyloid fibrils. A photoreactive Abeta(1-40) ligand was synthesized by substituting L-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Abeta(1-40) 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 Abeta 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 Abeta(1-5) F4Bpa tryptic peptide covalently attached to the C-terminal Abeta(29-40) 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 delta-methyl group of the Met35 side chain. The Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel alignment of Abeta peptides within amyloid fibrils.
Egnaczyk
G F
GF
Department of Pharmacology & Cell Biophysics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, P.O. Box 670575, Cincinnati, Ohio 45267-0575, USA.
Greis
K D
KD
Stimson
E R
ER
Maggio
J E
JE
D000143:Acids / Q000737:chemistry; D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels
D000143:Acids; D000682:Amyloid; D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D019920:Photoaffinity Labels; C078592:amyloid beta-protein (1-40)