Structural Features of the Aβ Amyloid Fibril Elucidated by Limited Proteolysis

Abstract

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 Abeta(1-40). In both reactions, the extreme N-terminal proteolytic fragment is released from fibrils as rapidly as it is from the Abeta 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 Abeta is not involved in the beta-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 Abeta(1-40) reaches plateau values corresponding to about 80% of the total available Abeta. This suggests that there are two classes of peptides in the fibril: while the majority of Abeta 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 Abeta 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 Abeta, while not directly involved in the beta-sheet network of the fibril, may contribute to fibril stability by participating in protofilament packing.

Conformational Abs recognizing a generic amyloid fibril epitope

“…In a recent characterization of the Ab response in mice injected with amyloid ␤ protein (A␤) fibrils, it was found that the majority of the Abs are directed at the N-terminal 12 residues of the peptide and are capable of crossreacting strongly with the monomeric peptide (7). This agrees well with the results of limited proteolysis studies of A␤ fibrils indicating an exposed, unstructured N-terminal region in the aggregate (8). Thus, such Abs tell us about those parts of the amyloidogenic peptide that are not involved in fibril structure but little about the nature of fibril structure itself.…”

“…A␤ peptides were solubilized and aggregated by a variation of the previously described protocol (8,20). Amyloid fibrils were grown from A␤(1-40) by incubating a 0.25 mg͞ml disaggregated solution of the peptide in PBS containing 0.05% sodium azide (PBSA) at 37°C together with a seed consisting of 0.1% by weight Abbreviations: PBSA, PBS containing 0.05% sodium azide; polyGln, polyglutamine; IAPP, islet amyloid polypeptide; A␤, amyloid ␤ protein; TTR, transthyretin; ␤2m, ␤2-microglobulin; ThT, thioflavin T.…”

“…Each peptide and protein required customized protocols for fibril formation. With the exception of polyGln aggregates, all amyloid fibrils, as well as collagen and elastin, were sonicated on ice with a probe sonicator for five consecutive 30-sec pulses before immobilization onto plastic microtiter plates.A␤ peptides were solubilized and aggregated by a variation of the previously described protocol (8,20). Amyloid fibrils were grown from A␤(1-40) by incubating a 0.25 mg͞ml disaggregated solution of the peptide in PBS containing 0.05% sodium azide (PBSA) at 37°C together with a seed consisting of 0.1% by weight Abbreviations: PBSA, PBS containing 0.05% sodium azide; polyGln, polyglutamine; IAPP, islet amyloid polypeptide; A␤, amyloid ␤ protein; TTR, transthyretin; ␤2m, ␤2-microglobulin; ThT, thioflavin T.…”

“…The results suggest the existence of a major conformational epitope present in many amyloid fibrils composed of diverse protein sequences. A␤ peptides were solubilized and aggregated by a variation of the previously described protocol (8,20). Amyloid fibrils were grown from A␤(1-40) by incubating a 0.25 mg͞ml disaggregated solution of the peptide in PBS containing 0.05% sodium azide (PBSA) at 37°C together with a seed consisting of 0.1% by weight of sonicated A␤ fibrils.…”

Orthogonal Cross-Seeding: An Approach To Explore Protein Aggregates In Living Cells

“…alternate aggregation routes or multistep assembly pathway(s), leading to multiple metastable intermediates (14)(15)(16)(17)(18)(19)(20). Current models propose that the species formed early in the course of aggregation, e.g., oligomers and protofibrils, are likely culprits in cytotoxicity and cell dystrophy (21)(22)(23)(24)(25).…”