Structural features of the A beta 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 th e amyloid folding motif. In the experiments reported here, we used trypsin and chymotrypsin to conduct limited proteolysis studies on synthetic amyloi d fibrils composed of the Alzheimer's disease peptide A beta (1-40). In bot h reactions, the extreme N-terminal proteolytic fragment is released from f ibrils 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 pos sess 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 networ k of the amyloid fibril, while the C-terminus is essentially completely eng aged in protective-presumably beta -sheet-structure. In both digests, relea se of the extreme N-terminal fragments of A beta (1-40) reaches plateau val ues 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 o f A beta molecules have an exposed N-terminus, about 20% of the peptides ha ve an N-terminus that is protected from proteolysis within the fibril struc ture. The most likely cause of this heterogeneity is the lateral associatio n of protofilaments into the fibril structure, which would be expected to g enerate a unique environment for those A beta N-termini located at protofil ament packing interfaces and/or in the interior core region between the pac ked protofilaments. This suggests that the N-terminal region of A beta, whi le not directly involved in the beta -sheet network of the fibril, may cont ribute to fibril stability by participating in protofilament packing.