Aβ(1–40) forms five distinct amyloid structures whose β-sheet contents and fibril stabilities are correlated

The aggregated, β-sheet rich amyloid structure represents a stable, alternatively folded state of polypeptides. Amyloid fibrils are associated with several important neurodegenerative diseases, such as Alzheimer’s and Huntington’s diseases 1, as well as a number of peripheral diseases of organ failure 2. Amyloid fibrils can be produced in vitro from many proteins, consistent with the polymeric structure of proteins and the relationship of amyloid fibrils to synthetic polymers 3. The fundamental unit of amyloid fibrils is the cross-β structure, in which β-sheet extended chains and sheet-sheet stacking interactions are perpendicular to the fibril axis and β-sheet H-bonds are parallel to the fibril axis 4. Details of the three-dimensional structures of amyloid fibrils are still being elucidated 5; 6; 7; 8; 9; 10; 11; 12 One striking feature of amyloid fibrils that sets them apart from most globular proteins is the ability of a single polypeptide chain to grow into more than one stable structure 13. The existence of multiple protein aggregate conformations, each of which can propagate with retention of structure, has long been speculated to be the basis for strain and species barrier effects in mammalian and yeast prion biology 14; 15. Polymorphism at the electron microscopy level, for example in Aβ amyloid fibrils 16; 17, has been known for some time, but it has not been clear whether these shape differences were due merely to different modes of super-assembly of a common protofilament structure, or to more substantial internal structural differences, such as β-sheet formation and side-chain packing. Previous solid state NMR and electron microscopy analyses suggest that the folded structures of Aβ(1–40) in two polymorphic amyloid fibrils are only modestly different, while the major structural differences are in how the folded peptides pack within the fibril cross-section 9; 18. Analysis of amyloid polymorphs of other protein sequences, however, suggests the possibility of polymorphic structures differing more extensively in the details of segmental folding, H-bonding and packing within the fibril 13; 19, and this is further suggested by the different manners in which sequence-related fragments from amyloid proteins pack within “β-spine” crystal structures 6. Polymorphism in amyloid fibrils may have profound biological consequences. It has been demonstrated that different polymorphic yeast prion fibrils generated in vitro produce different prion strain behavior when these are introduced into yeast 20; 21. Two structurally and functionally different polymorphic fibrils have been generated by exposing Aβ(1–40) to different growth conditions in vitro 22, and the identification of a third polymorphic form produced by elongation of Aβ peptides in vitro using fibrils extracted from AD brain 23 supports the idea that fibril polymorphism may contribute to variations within human diseases. In this paper we describe the creation of five self-propagating amyloid fibril structures by subjecting wild type Aβ(1–40) to different trial growth conditions. These Aβ(1–40) polymorphic fibrils vary considerably in structural properties. In particular, we find that amyloid polymorphs exhibit significant differences in the extent and locations of stable β-sheet, as probed by the number of backbone amides highly protected from hydrogen-deuterium exchange. We find that fibril stabilities assessed by their free energies of elongation 24 correlate extremely well with these β-sheet contents, consistent with the central role of β-sheet in fibril structure. The results illustrate the facility with which some peptides produce fibril polymorphs and suggest that β-sheet content contributes significantly to fibril properties.