Protein Motion and Configurations in a Form-Fitting Nanopore: Avidin in ClyA

We probe the molecular dynamics and states of an avidin protein as it is captured and trapped in a voltage-biased cytolysin A nanopore using time-resolved single-molecule electrical conductance signals. The data for very large numbers of single-molecule events are analyzed and presented by a new method that provides clear visual insight into the molecular scale processes. Avidin in cytolysin A has surprisingly rich conductance spectra that reveal transient and more permanently trapped protein configurations in the pore and how they evolve into one another. We identify a long-lasting, stable, and low-noise configuration of avidin in the nanopore into which avidin can be reliably trapped and released. This may prove useful for single-molecule studies of other proteins that can be biotinylated and then transported by avidin to the pore via their coupling to avidin with biotin-avidin linking. We demonstrate the sensitivity of this system with detection of biotin attached to avidin captured by the pore.