Characterization of Flagellar Filaments and Flagellin through Optical Microscopy and Label-Free Nanopore Responsiveness

In this study, we investigated the translocation characteristics of flagellar filaments (Salmonella typhimurium) and flagellin sub-units through silicon nitride nanopores in tandem with optical microscopy analysis. Even though untagged flagella are dark to the optical method, the label-free nature of the nanopore sensor allows it to characterize both tagged (Cy3) and pristine forms of flagella (including real-time developments). Flagella were depolymerized to flagellin subunits at ~65°C (most commonly reported temperature), ~70°C , ~75°C , and ~80°C to investigate the effect of temperature on depolymerization (Tdepol). The change in conductance (ΔG) profiles corresponding to Tdepol ~65°C and ~70°C were bracketed within the flagellin monomer profile whereas those of ~75°C and ~80°C extended beyond this profile, suggesting a change to the native protein state. The molecular radius calculated from the excluded electrolyte volume of flagellin through nanopore-based ΔG characteristics for each Tdepol of ~65°C ...