Abstract
The role of the polymorphism 129M/V in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. Little is however known about the molecular background to this phenomenon. We herein investigated the conformational stability, amyloid fibrillation kinetics and seeding propensity of different 129-mutants, located in β-strand 1 of PrP (129M (wt), 129A, 129V, 129L, 129W, 129P, 129E, 129K and 129C) in HuPrP90-231. The mutations 129V, 129L, 129K and 129C did not affect stability (midpoints of thermal denaturation, Tm =65-66 °C), whereas the mutants 129A and 129E and the largest side chain 129W were destabilized by 3-4 °C. The most destabilizing substitution was 129P which lowered the Tm by 7.2 °C. All mutants, except 129C, formed amyloid like fibrils within hours during fibril formation under near physiological conditions. Fibril forming mutants, showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants 129A, 129E, 129V, and 129L resembling the wild type 129M. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (129W), positive charge (129K) or rotational constraint (129P), the fibrillation was impeded. 129C did not form ThT/Congo red positive fibrils and non-reducing SDS PAGE of 129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 minutes after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness towards recruitment into the amyloid state.
Nystrom, Sofie