Formation of Critical Oligomers Is a Key Event during Conformational Transition of Recombinant Syrian Hamster Prion Protein

Abstract

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP 90 -232 ) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP 90 -232 showed an infrared absorbance spectrum typical of proteins with a predominant ␣-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3-2 M guanidine hydrochloride), the secondary structure of SHaPrP 90 -232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel ␤-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm ؊1 . Kinetic analysis of the transition process showed that the decrease in ␣-helical structures and the increase in ␤-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No ␤-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP 90 -232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP 90 -232 . A new model for the structural transition and aggregation process of the prion protein is described.The prion protein (PrP) 1 is, following the protein-only hypothesis, the sole agent causing a group of neurodegenerative disorders (1, 2), the so-called prion diseases or prionoses (3). The most important ones among them are bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and Creutzfeldt-Jakob disease in humans.The crucial step in transmission and manifestation of prion diseases is the conversion of benign monomeric cellular prion protein (PrP C ), which has a mainly ␣-helical secondary structure, to pathogenic multimeric scrapie prion protein (PrP Sc ), which is predominantly folded into ␤-sheets (4, 5). It is noteworthy that PrP C and PrP Sc do not differ in their amino acid sequence.Similar mechanisms play an essential role in a number of other neurodegenerative disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Therefore, the coupled processes of protein misfolding and aggregation, the kinetics of these processes, and the molecular species involved are of fundamental interest.Late products of the conversion are amyloid fibrils and amyloid plaques, which are widely considered to be direct effectors of the above mentioned disorders. However, evidence is accumulating that intermediates or by-product...

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

“…3A, inset). Such annular structures have been proposed to consist of a ring of eight monomers of PrP (40).…”

“…In this regard, several groups have studied the conformational transition of different recombinant prion protein constructs into ␤-rich aggregates (40,(42)(43)(44)(45)(46)(47)(48). Some studies pointed to the formation of ␤-rich oligomers during the conformational transition of PrP (40,48).…”

“…In this regard, several groups have studied the conformational transition of different recombinant prion protein constructs into ␤-rich aggregates (40,(42)(43)(44)(45)(46)(47)(48). Some studies pointed to the formation of ␤-rich oligomers during the conformational transition of PrP (40,48). It has been suggested that ␤-oligomers are not on the pathway to amyloid formation (42,46) and that the preference for forming either a ␤-oligomer or an amyloid aggregate can be dictated by the experimental conditions with pH Ͻ 5 and partially denaturing concentrations of urea (4 -5 M) favoring the ␤-oligomer and pH Ͼ 5 and low concentrations of urea (1-2 M) favoring amyloid (42).…”

Prion disease susceptibility is affected by β-structure folding propensity and local side-chain interactions in PrP

“…Later, Baskakov et al (17) provided evidence that the β-state was composed of oligomeric forms of PrP and suggested that these assemble after the PrP molecules completely unfold. Subsequent studies showed the β-structured oligomers to be predominantly octameric for both mouse and hamster PrP (18,19). Whether β-structured monomers of PrP exist is controversial.…”

Sensitive detection of aggregated prion protein via proximity ligation

“…2a). As the SP-PLA requires triple recognition events our observations suggest that denaturation with high concentrations of guanidine-HCl would induce incomplete disaggregation of PrP Sc as previously described, [29][30][31] with maintenance of at least trimers or small aggregates. No significant change in signal was detected upon denaturation of brain homogenate from uninfected hamster (Fig.…”