Several studies on scrapie in sheep have shown that the propensity for the conversion of a naturally occurring protein PrP from its normal cellular form (PrPC) to a virulent scrapie form (PrPSc), both in vitro and in vivo, correlates with susceptibility to the disease. Since scrapie results from the PrPC-to-PrPSc conversion, the PrPSc precursor occurring in PrP folding should play a key role in modulating disease occurrence. Susceptibility to classical scrapie correlates strongly with specific polymorphisms at positions 136, 154, and 171 in ovine prion proteins (ovPrPs). Therefore, I hypothesized that these polymorphisms affect scrapie susceptibility by modulating the structure and population of the PrPSc precursor. This hypothesis led to two sub-hypotheses: First, folding and unfolding of ovPrPs proceed through an intermediate. Second, this intermediate is the precursor of PrPSc.In this dissertation, I examined whether an intermediate state occurs in ovPrP (un)folding using a continuous-flow mixing method. Four PrP variants, comprising residues 94-233 of full-length ovPrP and correlated with differing susceptibilities to classical scrapie in sheep, were studied. An initial lag phase in refolding and unfolding kinetics indicates the presence of a native-like intermediate. I found that the relativepopulations and structural stability of the folding intermediates in these variants correlate with their propensities for classical scrapie. Variants susceptible to classical scrapie appear to have a larger population and higher structural stability in their intermediate states than do resistant variants. This could give susceptible variants more opportunities to undergo the PrPC-to-PrPSc conversion and oligomerize. Therefore, I argue that the observed folding intermediate is the precursor of PrPSc. A model for amyloid formation is proposed: Conformational folding of PrPC: N ↔ I ↔ U Assembly of PrPSc oligomers: (PrPSc)n + I → (PrPSc)n+1where N, I, and U represent native, intermediate, and unfolded states, respectively.Contrary to earlier studies on human PrP, I was unable to resolve any kinetic intermediate under refolding conditions within the dead-time of our instrument (~90 μs). Thus, I postulate that the folding kinetics of ovPrPs differs from the kinetics of human PrPs, which could reflect structural differences in their respective intermediates. Residues (136, 154, and 171) involved in genetic modulation are distant in the primary structure. Since ovPrP variants exhibit differing population and structural stability in their intermediate state, the polymorphism may modulate structural conversion through long-range interactions on the intermediate species. Consistent with this idea, a peptide model that is not involved in genetic modulation shows that local interactions in the vicinity of the disulfide bond do not suffice for the formation of a folding intermediate.
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