Model-Free Observation of Polypeptide Translocation Success Rate through a Nanopore

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Elucidating the motion of uniformly charged polymers in voltage-biased nanoscale channels has been a scientifically and technologically fruitful enterprise. Non-uniformly charged polymers such as polypeptides present a new set of challenges and opportunities, as they do not in general move through a nanopore unidirectionally or translocate the channel-containing membrane with unity probability. Here we demonstrate a single-molecule, model-free experimental technique to track the motion of a polypeptide in a channel and determine whether each polypeptide ultimately translocates through, or retracts from, the channel. The technique relies on the heterogeneity of the charge density along the polypeptide and its effect on the selectivity of the channel. In a nanopore under an electrolyte concentration gradient, the modulation of the channel selectivity is observable in real time as a change in the ionic conductance. For a polypeptide with different charge densities at the N- and C-termini, the ionic current at the end of a single molecule capture “event” reports on which end of the molecule exits the nanopore, and hence the direction of escape. As a demonstration, we report experimental observations of the interaction of a “diblock copolymer”-like neuronal intrinsically disordered protein, α-synuclein, with mitochondrial voltage-dependent anion channel (VDAC). The voltage-dependent translocation probability derived from the experiments shows that α-synuclein is bound to membrane surfaces with a distribution of binding energies that strongly depends on lipid species. These results have broad general implications for the interactions of peripheral proteins with lipid membranes.