| Abstract
| - In recent studies, we developed a protocol for in vitro conversion of full-length mouserecombinant PrP (Mo rPrP23−230) into amyloid fibrils [Bocharova et al. (2005) J. Mol. Biol. 346, 645−659]. Because amyloid fibrils produced from recombinant Mo PrP89−230 display infectivity [Legnameet al. (2004) Science 305, 673−676], polymerizatiom of rPrPs in vitro represents a valuable model forelucidating the mechanism of prion conversion. Unexpectedly, when the same conversion protocol wasused for hamster (Ha) rPrP23−231, we experienced substantial difficulties in forming fibrils. Whilesearching for potential reasons of our failure to produce fibrils, we probed the effect of methionine oxidationin rPrP. We found that oxidation of methionines interferes with the formation of rPrP fibrils and that thiseffect is more profound for Ha than for Mo rPrP. To minimize the level of spontaneous oxidation, wedeveloped a new protocol for rPrP purification, in which highly amyloidogenic Ha rPrP with minimallevels of oxidized residues was produced. Furthermore, our studies revealed that oxidation of methioninesin preformed fibrils inhibited subsequent maturation of fibrils into proteinase K-resistant PrPSc-likeconformation (PrP-res). Our data are consistent with the proposition that conformational changes withinthe central region of the protein (residues 90−140) are essential for adopting PrP-res conformation anddemonstrate that methionine oxidation interferes with this process. These studies provide new insightinto the mechanism of prion polymerization, solve a long-standing practical problem in producing PrP-res fibrils from full-length PrP, and may help in identifying new genetic and environmental factors thatmodulate prion disease.
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