Structural Investigations of the Prion Protein het-s

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DESCRIPTION (provided by applicant): The "protein-only hypothesis" states that prion diseases such as scrapie in sheep, bovine spongiform encephalopathy (BSE) and Creutzfeldt-Jakob disease in human are distinct from infectious diseases caused by bacteria, viruses, or viroids, in that the origin of the disease is related to conformational alterations of an ubiquitous protein and that nucleic acids are not essential for the propagation of the infectious agent. Thus, prions are infectious proteins which propagate by converting the normal form of the protein into an altered beta-sheet-rich conformation. Prion proteins have also been identified in lower eukaryotes, namely yeast and the filamentous fungus Podospora anserina. Totally, there are only four different proteins known so far which may adopt a prion state, and only for the het-s prion system in Podospora anserina has it been shown convincingly that the het-s prion protein is indeed the infectious agent. Furthermore, the het-s protein is the only known prion protein of which the prion state, pHET-s, is part of a normal cellular function, namely cell fusion incompatibility, a common feature in filamentous fungi. The aim of the project described in this proposal is to get structural insights into the components of the het-s prion system of the filamentous fungus Podospora anserina using solution-state nuclear magnetic resonance spectroscopy (NMR), other biophysical techniques, mutagenesis and in vivo studies. The three-dimensional structures of the non-prion form of the het-s protein and a prion-incompetent analog will be determined and accompanied with mutagnesis to elucidate the spatial regions and the residues important for the generation of infectivity. Furthermore, structural studies of the prion form of the het-s protein will be initiated to get insights into the conformational transition of the het-s protein which has been assoicated with infectivity. The structural knowledge of the individual components and detailed analysis of the conformational transition which is associated with the generation of a prion phenotype will therefore extend our understanding of the het-s system in particular, and also of the mechanism of prions and their origin of infectivity in general.