Identification and removal of proteins that co‐purify with infectious prion protein improves the analysis of its secondary structure

Transmissible spongiform encephalopathies (TSEs), or prion diseases are fatal, neurodegenerative diseases that include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in deer and elk and bovine spongiform encephalopathy in cattle (1). The conformational conversion of soluble, protease-sensitive mammalian prion protein (PrPC) from a structure rich in α-helix to an insoluble, protease-resistant form that is rich in β-sheet (PrPSc) is a critical event in prion disease pathogenesis (2). Prion diseases are unique among protein misfolding disorders in that they are transmissible (1). PrPSc is believed to be the primary component of the prion infectious agent and the molecular basis of prion diseases is likely to be at least in part determined by specific PrPSc conformations (3–7) which lead to different prion strains exhibiting characteristic phenotypes in vivo (2;8). In order to understand how PrPSc conformation may influence disease phenotype in vivo, it is important to determine the structure of PrPSc from different prion strains. The structural differences between naturally occurring PrPC and PrPSc are remarkable, given that they share the same primary amino acid sequence. The structure of PrPC has been studied in detail by high resolution NMR spectroscopy (9–11) and, to a lesser extent, by X-ray crystallography (12). These data cumulatively show that PrPC is approximately 47% α-helical, 3% β-strand and contains a natively unstructured N-terminus (9). By contrast, because PrPSc is highly aggregated and difficult to purify to homogeneity, no high resolution structures have been determined. Lower resolution techniques such as circular dichroism (13), fourier-transform infrared spectroscopy (FTIR) (14–16), and hydrogen-deuterium exchange (17) have established that PrPSc aggregates contain much more β-sheet secondary structure compared to monomeric PrPC. Most of the current literature suggests that PrPSc retains some portion of its pre-conversion α-helix content when converted to PrPSc (2;18;19), although other studies suggest that PrPSc retains no α-helix character at all (13;16;17). These discrepancies could be explained by the presence of contaminating proteins that co-purify with PrPSc. Various protocols for PrPSc enrichment developed over the last three decades (20–24) have yielded PrPSc samples which are actually complex mixtures that can contain carbohydrates (25), lipids (26), nucleic acids (27), other proteins (28;29), and probably metal ions (30). However, no structural study has directly addressed how these contaminants might contribute to structural features attributed to PrPSc. Using an improved PrPSc purification protocol, we have examined the secondary structure of mouse and hamster PrPSc both before and after the removal of major protein contaminants that commonly co-purify with PrPSc. Tandem mass spectrometry (LC-MS/MS) was used to identify the proteins in our samples while FTIR was used to probe secondary structure. The results show that non-PrP proteins do contribute to absorbance associated with α-helical, loop, turn and β-sheet structures that previously have been attributed solely to PrPSc. In particular, ferritin, which is known to consist almost exclusively of α-helices (31) and which we previously identified as the most abundant contaminant in enriched PrPSc samples (29), absorbed in the α-helix region (1652cm−1) of the spectrum. However, even after many non-PrP proteins were removed and ferritin was reduced by 100 fold, absorbance at 1652cm−1 was still detectable. Thus, while contaminating proteins do alter the FTIR spectrum attributed to PrPSc, the secondary structure remaining after their removal is likely derived from PrPSc itself suggesting that PrPSc contains some α-helical, loop, and turn secondary structure.