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...
Amyloid protofibril formation of phosphoglycerate kinase (PGK) and Syrian hamster prion protein (SHaPrP(90-232)) were investigated by static and dynamic light scattering, size exclusion chromatography and electron microscopy. Changes in secondary structure were monitored by Fourier transform infrared spectroscopy and by circular dichroism. Protofibril formation of the two proteins is found to be a two-stage process. At the beginning, an ensemble of critical oligomers is built up. These critical oligomeric states possess a predominant beta-sheet structure and do not interact considerably with monomers. Initial oligomerization and transition to beta-sheet structure are coupled events differing in their details for both proteins. Intermediate oligomeric states (dimers, trimers, etc.) are populated in case of PGK, whereas SHaPrP(90-232) behaves according to an apparent two-state reaction between monomers and octamers rich in beta-structure with a reaction order varying between 2 and 4. All oligomers coalesce to PGK protofibrils in the second stage, while SHaPrP(90-232) protofibrils are only formed by a subpopulation. The rates of both growth stages can be tuned in case of PGK by different salts preserving the underlying generalized diffusion-collision mechanism. The different kinetics of the early misfolding and oligomerization events of the two proteins argue against a common mechanism of protofibril formation. A classification scheme for misassembly mechanisms of proteins based on energy landscapes is presented. It includes scenarios of downhill polymerization to which protofibril formation of PGK and SHaPrP(90-232) belong.
Classical genetic analysis is not possible with the protist Plasmodiophora brassicae due to the intracellular life of this obligate biotrophic parasite. An electrophoretic karyotype has been obtained using contour‐clamped homogeneous electric field gel electrophoresis to facilitate gene mapping of P. brassicae. Using two different separation conditions 16 chromosomal bands of P. brassicae were distinguished ranging in approximate size from 2.2 Mb to 680 kb. According to this determination of chromosome number and size, the total genome size of P. brassicae was estimated to be 20.3 Mb. The chromosomal bands were further designated by their hybridization pattern with repetitive elements of P. brassicae. The repetitive element H4 (1800 bp) hybridized with 14 chromosomal bands, but the sequence of H4 showed no homology to known centromere or telomere structures and revealed no repetitive motifs.
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