Existing biological nanopores are often too narrow to accommodate large proteins and biomolecular complexes in their native folded states, precluding the analysis of megadalton assemblies. Here, we report the self-assembly of the cholesterol-dependent cytolysin Perfringolysin O (PFO) into stable transmembrane nanopores composed of 46 {+/-} 9 monomers, with an inner pore diameter of 28.5 {+/-} 5.6 nm and a length of 9.8 nm. We demonstrate that despite the large size of the pore assembly, PFO pores exhibit a stable open-pore current with a high signal-to-noise ratio, making them suitable for resistive-pulse recordings. Moreover, PFO nanopores enable accurate, calibration-free, and label-free sizing of individual proteins, multi-protein complexes, and viral particles across a large molecular-weight range spanning 50 kDa to 3.4 MDa. The exceptionally large diameter of these pores enables, for the first time, resistive-pulse-based characterization of intact virus particles and ribosomes with a biological nanopore. Specifically, we determined the volume, shape, and diameter of complete capsids of recombinant adeno-associated virus serotype 2 (rAAV2) as well as capsid fragments. Finally, simultaneous analysis of molecular volume and shape resolved intact 70S ribosomes from their dissociated 30S and 50S subunits in a mixture. By extending biological nanopore sensing to single-particle characterization of large protein complexes well beyond the reach of existing pores, this approach introduces PFO nanopores as a versatile platform for label-free, single-particle analysis in solution.
Vracar, A., Salyahetdinova, V., Balog, S., Ianiro, A., Mukhopadhyay, A., Mayer, M.
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