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...
The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross-beta structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-state-->fibril type I-->fibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.
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