Our purpose is to determine structural features, and the factors influencing those features, of the soluble cellular form of the prion protein (PrPC) as well as the insoluble form (PrPSc). This should enlighten our understanding of the mechanism of conversion of PrPC to PrPSc. Nuclear magnetic resonance (NMR) will be the principal tool utilized to investigate the structures of peptides and protein fragments from prion proteins and the interconversions of different structures in response to external conditions and by selected mutations. For soluble peptides and protein fragments, high resolution solution NMR techniques will be used to elucidate the structure. Recent results here make us cautiously optimistic that either the whole prion protein or very large domains (e.g., the 90-231 fragment) will be available via recombinant DNA techniques for the solution NMR studies. While study of the whole protein or such a large moiety is most desirable, the examination of smaller synthetic fragments can still be enlightening, in particular in the context of other experimental and computational information. Fully 13C/15N-labeled prion protein can be obtained readily via efficient cloning and expression, and should be extremely valuable for the solution NMR studies. Peptides with isotope labels in specific locations can also be synthesized (see Peptide Synthesis Project); while these can be utilized for solution NMR studies, they are essential for the solid state NMR aspects of this project. For insoluble forms of the peptides and protein fragments, solid state NMR methods will be employed. In particular, rotational resonance and other solid state NMR dipolar experiments will be employed to determine distances in selectivity 13C-, 15N-, or 19F-labeled peptides. Additional conformational characterization can be done by interpreting 13C chemical shifts. In small peptide models, systematic searches of both intra- and intermolecular distances will be done. For larger peptide fragments this may not be feasible, so we will introduce paramagnetic centers to obtain longer range, less precise distances to generate initial structural models. These will then be refined and checked by further use of local distance measurements with rotational resonance or heteronuclear and homonuclear REDOR-based experiments. The combination of solution and solid state data will be interpreted to try to understand the conformational changes leading to the formation of PrPSc.
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