Mixed Company in a Protein Nanopore: Transient Double Occupancy Enables Direct Exchange of Pore Ligand in Nanopore-Based Single Molecule Sensing

Abstract

Partitioning of poly(ethylene-glycol) (PEG) into an alpha-hemolysin nanopore gives rise to long-lasting blockades (τ≈0.1-10 ms) of ionic current. The depth of blockade shows exquisite sensitivity for polymer length, yielding mass spectra with single monomer resolution in the range between MW≈700-2200 or 15-50 repeat units (r.u.). Unexpectedly, high-resolution recordings of single PEG blocking events using a mixture of two monodisperse species unequivocally identified direct transitions between levels corresponding to 28 and 32 r.u. without an intervening unblocked interval. Closer analysis revealed that these occur by the intermediary of shorter, deeper blocks. Based on statistics and current amplitude distributions, we are able to identify three such deeply blocked states, each corresponding to the simultaneous presence in the pore of two ligands: either 2xPEG-28, 2xPEG-32 or PEG-28+PEG-32 (mixed double occupancy). Direct PEG28->PEG32 transitions (or vice versa) are observed only with an intervening mixed occupancy block, which, however, can also result in return to the first blocked level. We conclude that the alpha-hemolysin pore is capable of accomodating two PEG oligomers, with ligand exchange occurring by displacement and translocation of the first blocker.View Large Image | View Hi-Res Image | Download PowerPoint Slide