The properties of PrP and its low abundance in brain have made it very difficult to isolate sufficient amounts of the cellular or scrapie isoforms to undertake high resolution structural studies. PrP 27-30, the most easily purified form, is heterogeneous and insoluble, thus is not suited to crystallization and X-ray diffraction or analysis by NMR. Recombinant forms of the protein may soon be available but these will require stable isotope incorporation for NMR analysis. Existing methods allow expression of recombinant proteins with heavy isotopes incorporated in all amino acids of one type. NMR on solid phase samples however requires specific incorporation of isotope labels at selected sites that can participate in magnetization exchange using techniques such as rotational resonance. Chemical synthesis provides an alternative strategy to recombinant over-expression but the construction of even the 142 amino acid fragment corresponding to PrP 27-30 is beyond the scope of current methodology. A combination of chemical synthesis of peptides and enzymatic ligation does however offer a realistic prospect of obtaining sufficient quantities of pure, homogeneous, protein. Funding has been provided for a one-year project to develop the synthesis of the 142 amino acid PrP fragment corresponding to residues 90-231. Preliminary results are encouraging and it is very probable that this will be completed within the coming year. In the following year we shall refine and improve the yield of this synthesis. We shall also design and synthesize isotopically labelled analogs containing only two labelled sites for solid state NMR. As an adjunct to making entirely synthetic peptides, we shall develop protocols for the ligation of synthetic peptides to larger recombinant PrP fragments. Recent results on the expression and purification of a large recombinant PrP fragment have been very encouraging. The peptide ligations and the recombinant expression will be part of a continuing collaboration between UCSF and scientists at Genentech. Thus, using a combination of approaches we shall obtain samples suitable for NMR studies in both the liquid and the solid states. Subsequently these studies will be extended to molecules containing known pathogenic mutations. We shall also synthesize analogs containing other groups such as fluorine atoms and spin labels for other NMR measurements. We shall develop the synthesis of PrP analogs containing regions of constrained structure which force the molecules to adopt certain conformations which may be particularly helpful in determining the features of PrP that contribute to the conversion of PrPC to PrPSc. All the synthetic species will be subjected to a wide variety of refolding conditions selected to encourage the development of PrPSc. They will be assayed for prion infectivity in Syrian hamsters or in appropriate transgenic hosts. The latter will be selected to minimize species barriers due to differences in the primary sequences. In the event that infectivity can be generated, we shall determine the minimum regions of beta-sheet required for pathogenicity. The most significant of these species will then be isotopically labelled for structural studies by NMR. The refolding of all these species will also be studied in conjunction with structural analyses by techniques that will include CD, FTIR and fluorescence spectroscopy, as adjuncts to the NMR studies.
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