Molecular aspects of copper and zinc promoted prion-prion interactions

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DESCRIPTION (provided by applicant): Despite intense effort, the precise function of the prion protein remains unclear as well as the mechanism of prion accumulation and neuronal damage. Recent studies now indicate that prion self-recognition may be an important factor in both the normal function and misfunction of this protein. Prion has also been identified as a metalloprotein, binding multiple coppers and possibly zinc. The binding of these cofactors appears to act as a switch that induces prion-prion interactions. We have developed fluorescently labeled peptide models derived from the metal binding region of the prion protein that allow prion-prion interactions and metal binding to be investigated on the molecular level. These models are anchored to a membrane surface so as to mimic the prion protein's cellular environment. To address the specific aim of determining the structural elements responsible for prion-prion interactions, peptide models will be investigated as a function of added copper and zinc using pulsed-field gradient NMR and fluorescence techniques. The second specific aim of determining the copper binding mechanism and the relevance of different copper loaded states will be addressed using circular dichroism, electron paramagnetic resonance and metal catalyzed oxidation reactions in conjunction with tandem mass spectrometry. All experimental studies will be enhanced with a parallel set of molecular modeling studies. Results from the aims in this proposal will provide insight into the signaling mechanism for prion endocytosis and the means by which it might transduce other signals. In addition, these studies are anticipated to provide information on how prion-prion interactions may lead to disease causing prion mis-folding. All aspects of this research project are designed to be conducted by undergraduate and masters-level students. They should become proficient in peptide synthesis, purification and characterization plus a variety of cutting-edge instrumental techniques. They will also be introduced to computational methods of data analysis and molecular modeling.