Structural features of the Aβ amyloid fibril elucidated by limited proteolysis

Although the gross morphology of amyloid fibrils is fairly well understood, very little is known about how the constituent polypeptides fold within the amyloid folding motif. In the experiments reported here, we used trypsin and chymotrypsin to conduct limited proteolysis studies on synthetic amyloid fibrils composed of the Alzheimer's disease peptide A beta (1-40). In both reactions, the extreme N-terminal proteolytic fragment is released from fibrils as rapidly as it is from the A beta monomer, while other proteolytic fragments are generated much more slowly. Furthermore, aggregated material isolated by centrifugation of intermediate digestion time points from both proteases contains, in addition to full-length material, peptides that possess mature C-termini but truncated N-termini. These data strongly suggest that the N-terminal region of A beta is not involved in the fi-sheet network of the amyloid fibril, while the C-terminus is essentially completely engaged in protective-presumably beta -sheet-structure. In both digests, release of the extreme N-terminal fragments of A beta (1-40) reaches plateau values corresponding to about 80% of the total available A beta. This suggests that there are two classes of peptides in the fibril: while the majority of A beta molecules have an exposed N-terminus, about 20% of the peptides have an N-terminus that is protected from proteolysis within the fibril structure. The most likely cause of this heterogeneity is the lateral association of protofilaments into the fibril structure, which would be expected to generate a unique environment for those A beta N-termini located at protofilament packing interfaces and/or in the interior core region between the packed protofilaments. This suggests that the N-terminal region of A beta, while not directly involved in the beta -sheet network of the fibril, may contribute to fibril stability by participating in protofilament packing.