Atomic force microscopy to characterize the molecular size of prion protein

The conformational transition of α-helix-rich cellular prion protein (PrPC) to an isomer with high β-sheet content is associated with transmissible spongiform encephalopathies. With the ultimate long-term goal of using imaging techniques to study PrP aggregation, we report the results of initial experiments to determine whether PrP molecules could be visualized as single molecules, and if the observed size corresponded to the calculated size for PrP. The investigation of single molecules, and not those embedded into larger aggregates, was the key in our experimental approach. Using atomic force microscopy (AFM) as an imaging method, the immobilization of recombinant histidine (His)₁₀-tagged PrP on mica was performed in the presence of different heavy metal ions. The addition of Cu²⁺ resulted in an enhanced PrP immobilization, whereas Ni²⁺ reduced coverage of the surface by PrP. High-resolution data from dried PrP preparations provided a first approximation to geometrical parameters of PrP precipitates, which indicated that the volume of a single PrP molecule was 30 nm³. Molecular dynamics simulations performed to complement the structural aspects of the AFM investigation yielded a calculated molecular volume of 33 nm³ for PrP. These experimentally observed and theoretically expected values provide basic knowledge for further studies on the size and composition of larger amyloidal PrP aggregates, PrP isoforms or mutants such as PrP molecules without octarepeats.