Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity

Abstract

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPsC) and a component of the infectious scrapie agent; the potential to form amyloid is a result of a posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 "C, 100 "C, and 132 "C for 30 min did not change the @-sheet secondary structure; the infectivity slightly diminished at 132 "C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solventexposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, @-pleated sheet-like secondary structure component, an increasing amount of a-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the @-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the @-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.

Mechanisms of prion protein assembly into amyloid

“…1). Proteinase K (PK) digests PrP C completely but cleaves PrP Sc specifically at residue 89 or 90 leaving the C terminus (amino acids 90-231) intact; this protease-resistant fragment is denoted PrP 27-30 and is fully infectious (7)(8)(9)(10). If PK digestion is carried out in the presence of detergents, PrP 27-30 assembles into prion rods, which have the tinctorial properties of amyloid (11,12).…”

A role for intermolecular disulfide bonds in prion diseases?

“…For example, scrapie infectivity seems to survive extremely high temperatures (25), which is more unusual for a polymer stabilized by only noncovalent interactions than for a polymer stabilized by both covalent bonds and (protective) noncovalent interactions. The cellular form PrP C also has a remarkable conformational stability in the vicinity of the disulfide bond (21) and an unusually fast folding time (26), which may derive from the need to stabilize the critical intracellular disulfide bond against thiolate attack.…”

The Role of Prion Peptide Structure and Aggregation in Toxicity and Membrane Binding

“…However, the protease-resistant core of PrP SC is mostly ␤-sheet (43%) with less ␣-helical structure (30%), and it has a tendency to polymerize into amyloid fibrils (Prusiner et al, 1983;Pan et al, 1993;Safar et al, 1993;Baldwin et al, 1994). Based on these structural measurements, researchers have postulated that PrP SC formation involves a conformational transformation resulting in increased ␤-sheet content (Pan et al, 1993;Safar et al, 1993;Baldwin et al, 1994).Structural changes similar to the PrP C to PrP SC conformational conversion associated with prion diseases are typical in amyloidoses such as Alzheimer's disease, primary systemic amyloidosis, familial amyloid polyneuropathy, and type II diabetes (Gabizon and Prusiner, 1990;Sipe, 1992;Kelly, 1996;Taubes, 1996;Lansbury, 1999). Both the amyloidoses and the prion diseases are thought to result from the conversion of normally soluble and functional proteins into abnormal ␤-sheet-rich structures that have propensities to fibrilize (Colon and…”

“…Fourier transform infrared spectroscopy and circular dichroism (CD) demonstrate that PrP C is predominantly ␣-helical (42%) with little ␤-sheet structure (3%). However, the protease-resistant core of PrP SC is mostly ␤-sheet (43%) with less ␣-helical structure (30%), and it has a tendency to polymerize into amyloid fibrils (Prusiner et al, 1983;Pan et al, 1993;Safar et al, 1993;Baldwin et al, 1994). Based on these structural measurements, researchers have postulated that PrP SC formation involves a conformational transformation resulting in increased ␤-sheet content (Pan et al, 1993;Safar et al, 1993;Baldwin et al, 1994).…”