Abstract
Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C1/2 value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C1/2 value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-beta core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core.
Highlights
Besides the native globular shape, the vast majority of proteins and peptides are capable of forming alternative, well defined structures with a specific cross- sheet fold referred to as amyloid fibrils [1]
We avoided replacing the residues that are known to be “functionally” important for PrPSc propagation, such as the residues involved in the dominant-negative effect [20], the residues that account for the species-species barrier, or those associated with familial forms of prion disease [21]
Because in PrPC glycans and the glycosylphosphatidylinositol anchor are attached to asparagine 196 and serine 230, respectively, we assumed that labeling prion protein (PrP) at these residues should have minimal steric impact on fibril formation
Summary
PrP, full-length recombinant prion protein; PrPC, cellular isoform of the prion protein; PrPSc, disease associated isoform of the prion protein; WT, wild type; PK, proteinase K; GdnHCl, guanidine hydrochloride; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; ThT, thioflavin T; FTIR, fourier transform infrared. We have described a reliable approach for elucidating the global and site-specific conformational stability of amyloid fibrils. We employed this method to elucidate the conformation of fibrils produced from the full-length mouse PrP. We found that within the amyloid fibrillar structure, PrP polypeptides adopt two independent folding domains. These domains are characterized by different C1⁄2 values in GdnHCl-induced denaturation. The denaturation profile of the most stable domain was superimposable with the global unfolding of amyloid structure This domain appears to form cross-sheet fibrillar core, whereas the less stable domain might be responsible for forming interfaces between filaments. Our studies established a new technique for studying amyloid fibrils and provided an important new insight into the substructure of PrP fibrils
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