Pneumolysin nanopores with 20 nm inner diameter to characterize the size and shape of Tau oligomers

Abstract Protein nanopores are emerging as versatile tools to fingerprint biomolecules due to their capability to characterize single molecules without the requirement for labeling. A long-standing challenge with biological nanopores is, however, that large biomolecules in their native state are often too large to enter these pores. Here, we report the self-assembly of approximately 35 ± 5 pneumolysin (PLY) toxins to a stable transmembrane pore with a diameter of 20 ± 3 nm, an effective length of 9.5 nm, and excellent low noise characteristics in the context of nanopore-based resistive pulse recordings. The exceptionally large pore diameter enables the characterization of the size and shape of individual proteins and protein complexes ranging in molecular weight from 50 kDa to 0.8 MDa. Moreover, PLY pores make it possible to follow the time course of the formation of oligomers of tau protein in solution by revealing the size, monomer number, approximate shape, and abundance of these oligomers. At least four characteristics make PLY pores well suited for the characterization of heterogeneous amyloid oligomer samples: First, they are not prone to clogging. Second, they provide label-free single particle analysis. Third, their large diameters make it possible to characterize a wide range of amyloid oligomer sizes with high resolution. And fourth, resistive pulse recordings from these pores provide stable open pore current baselines with low electrical noise. Competing Interest Statement The authors have declared no competing interest.