The global objective of this proposal is to elucidate, at the molecular level, the mechanism and function of copper binding in the prion protein and the relationship of this interaction to the propagation of prion mediated neurological disease. The prion protein (PrP) is responsible for a class of fatal dementing diseases called the transmissible spongiform encephalopathies (TSEs) which include mad cow disease and the human affliction Creutzfeldt-Jakob disease. PrP is a globular, membrane-bound, glycoprotein found in all mammals and avian species. Despite nearly twenty years of research on this remarkable protein, its physiological function has been unclear. Recent work, however, demonstrates that the flexible N-terminal domain of PrP binds copper ions cooperatively and with high affinity. Physiological studies now suggest that PrP plays a crucial role in copper homeostasis within the central nervous system. This is an exciting development given the current interest in biological mechanisms of copper trafficking and recent suggestions into the interplay between improper metal ion regulation and neurological disease. in 2000, the PI's laboratory reported detailed spectroscopic measurements that revealed the molecular features of the copper binding sites within the PrP N-terminal domain. The goal of the proposed research is to further clarify the molecular features of the copper binding site, evaluate PrP function and determine how copper binding participates in the TSEs. The three specific aims will use multiple biophysical and spectroscopic approaches.
Aim #1 focuses on extending current spectroscopic and structural work to the full-length, recombinant protein. This work will take advantage of new protein ligation chemistries for placing isotopic labels within the N-terminal domain.
Aim #2 focuses on the possibility that PrP's function is to transport copper through endocytosis. These studies will explore how PrP's structure responds to pH and copper concentration.
Aim #3 will examine the interplay between copper binding and conversion of PrP to the beta-sheet rich form implicated in prion disease. These studies will explore the effects of mutations and other sequence features that confer predisposition to the TSEs. Taken together, this research plan promises to significantly advance the understanding of PrP function and the molecular mechanisms of TSE propagation.
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