Abstract
Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.
Highlights
More than 15 severe maladies, including prion, Alzheimer, and Parkinson diseases, are related to the formation of specific -sheet-rich protein aggregates known as amyloid fibrils [1, 2]
After screening a variety of experimental conditions, we found that the proteinase K (PK)-resistant core elongated substantially upon brief exposure of the fibrils to 80 °C in the presence of 1% Triton X-100 (Fig. 1a, compare lanes 2 and 3)
Temperature-induced extension of the PK-resistant core was strictly limited to the amyloid fibrils; rPrP converted into other forms, such as ␣-helical monomers (␣-rPrP) or -sheet-rich oligomers, failed to display annealing (Fig. 1a)
Summary
More than 15 severe maladies, including prion, Alzheimer, and Parkinson diseases, are related to the formation of specific -sheet-rich protein aggregates known as amyloid fibrils [1, 2]. Recent studies demonstrated that a broad range of proteins unrelated to any known conformational disease are capable of forming -sheet-rich amyloid forms both in vitro and in vivo [3,4,5,6]. This finding has led to the proposition that the ability to fold into amyloid structures is not a unique property of certain proteins associated with degenerative maladies; rather, it may well be a feature of polypeptides in general [7]. Our studies provide a novel insight into the maturation and evolution of amyloid structures
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