Over Two Million Protein Translocations Reveal Optimal Conditions for High Bandwidth Recordings, Studying Transport Energetics, and Electric-Field Unfolding

The gradual tapered geometry of nanopipettes offers a unique perspective on protein transport through nanopores since both a gradual and fast confinement is possible depending on translocation direction. Protein capture rate, unfolding, speed of translocation, and clogging probability are studied by toggling the lithium chloride concentration between 4 M and 2 M. Interestingly, the proteins in this study could be transported with or against the electrophoresis and offer vastly different attributes of sensing and affect whether a protein unfolds during pore transit. A ruleset for studying proteins is developed that prevents irreversible pore clogging and yielded upwards of >100,000 events/nanopore. Minimizing clogging also permitted higher quality data via the use of smaller pores (i.e., <2x the size of the protein) including higher SNR recordings and data acquisition at the highest available bandwidth (100 kHz). The extended duration of experiments further revealed that the capture rate takes ~2 hours to reach a steady state with a value ~3x greater than the initial reading, emphasizing the importance of reaching equilibrated transport for studying the energetics of protein transport (i.e., diffusion vs barrierlimited). Even in the equilibrated transport state, improper lowpass filtering was shown to distort the classification of diffusion-limited vs barrier-limited transport. Finally electric-field induced protein unfolding was found to be most prominent in EO dominant transport whereas EP dominant events show no evidence of unfolding. Thus, our findings showcase the optimal conditions for protein translocations and the impact on studying protein unfolding, transport energetics, and acquiring high bandwidth data.