| Abstract
| - Although the gross morphology of amyloid fibrils is fairly well understood, very little is knownabout how the constituent polypeptides fold within the amyloid folding motif. In the experiments reportedhere, we used trypsin and chymotrypsin to conduct limited proteolysis studies on synthetic amyloid fibrilscomposed of the Alzheimer's disease peptide Aβ(1−40). In both reactions, the extreme N-terminalproteolytic fragment is released from fibrils as rapidly as it is from the Aβ monomer, while other proteolyticfragments are generated much more slowly. Furthermore, aggregated material isolated by centrifugationof 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 theN-terminal region of Aβ is not involved in the β-sheet network of the amyloid fibril, while the C-terminusis essentially completely engaged in protectivepresumably β-sheetstructure. In both digests, releaseof the extreme N-terminal fragments of Aβ(1−40) reaches plateau values corresponding to about 80% ofthe total available Aβ. This suggests that there are two classes of peptides in the fibril: while the majorityof Aβ molecules have an exposed N-terminus, about 20% of the peptides have an N-terminus that isprotected from proteolysis within the fibril structure. The most likely cause of this heterogeneity is thelateral association of protofilaments into the fibril structure, which would be expected to generate a uniqueenvironment for those Aβ N-termini located at protofilament packing interfaces and/or in the interiorcore region between the packed protofilaments. This suggests that the N-terminal region of Aβ, while notdirectly involved in the β-sheet network of the fibril, may contribute to fibril stability by participating inprotofilament packing.
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