Atomic Resolution Insights into the Aggregation of the Murine Prion Protein by NMR

Abstract

The aggregation mechanism of the prion protein is known to be complex. It is highly heterogeneous since multiple oligomeric as well as fibrillar structures are being populated. It is essential to understand the disease-associated assembly of prion protein monomers into high-molecular-weight polymers in mechanistic terms in order to be able to develop tailored therapeutic approaches. This requires following the aggregation process in detail on the molecular level. Kinetic methods are especially suited in this respect as the desired mechanistic information is contained in the kinetic data. We have investigated the aggregation of the wildtype murine prion protein at atomic resolution using kinetic two-dimensional (2D) NMR spectroscopy. We have found amino-acid-specific differences for the decrease in the monomer concentration thereby delineating heterogeneity in the aggregation kinetics. SDS PAGE shows that oligomers of differing size are being formed which are characterized by extremely high stability. Furthermore, Congo red dye binding experiments demonstrate that the prion protein assembles into amyloid-like fibrils. By combining the data acquired by using different methods we derive a model for the aggregation mechanism of the prion protein. In addition, we will also present kinetic 2D NMR data on the aggregation of disease-associated single point mutants of the prion protein such as V180I and E200K and discuss which aggregation sub-steps are affected by the mutations.