The major goals for this project are the structural elucidation of soluble PrP fragments using NMR spectroscopy. It is clear from both biological and preliminary studies that the region of the PrP/c molecule that lie close to the N-terminus are potentially of great significance in the structure and function of the PrP, and in the alpha-->beta conversion that is thought to be the source of prion disease and infectivity. The first specific aim will concentrate on the determination of the structure of the H1 and octarepeat regions of the SHaPrP/c protein, in the context of sizeable fragment containing regions that are known to be folded in solution. Similarity between the NMR spectra of the full-length construct 29-231 and a shorter fragment 90-231 indicate that the longer fragment may have a domain structure, but a significant increase in stability is also indicative that the H1 and octarepeat regions have structural significance. Binding of metal ions of possible physiological significance will be investigated. Preliminary 3D triple resonance NMR spectra of the 29-231 fragment are excellent, and give great promise that resonance assignments and solution structure determination will be possible in this challenging system. It is of considerable importance and interest to discover the structural basis for the alpha-->beta conversion. To this end, the second and third specific aims will address the structure of wt and mutant chimeric MH2M PrP fragments, point mutants A117V and P102L and a triple A113V, A115V, A118V mutant. The mutant residues are in the center of the region that is vital to the conformational plasticity of the N- terminal section of PrP, and the point mutants have been implicated in familial prion disease. Structural studies on these proteins should allow us to probe the nature of the alpha-->beta conversion. Certainly the change of three helix-promoting Ala residues for three beta-promoting Val residues would be expected to favor the veta form of the protein. Circular dichroism studies of the 90-231 recombinant triple mutant show substantial beta-sheet structure and much less alpha-helix. In an exciting new development, a 106-residue PrP fragments has been prepared where several loops of the intact protein have been deleted. This fragment is the first soluble PrP/sc-like molecule and represents our best opportunity to learn about the structure of the disease-causing isoform. The fourth specific aim therefore addresses structure determination by NMR of this fragment.
The specific aims are closely related to each other and to the goals of the overall program project. They address specifically the structural basis of the formation of prions by structure elucidation of folded fragments in solution.
