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 subunits 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 similar to 65 degrees C (most commonly reported temperature), similar to 70 degrees C, similar to 75 degrees C, and similar to 80 degrees C to investigate the effect of temperature (T-depol) on depolymerization. The change in conductance (Delta G) profiles corresponding to T-depol similar to 65 degrees C and similar to 70 degrees C were bracketed within the flagellin monomer profile whereas those of similar to 75 degrees C and similar to 80 degrees 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 Delta G characteristics for each T-depol of similar to 65 degrees C, similar to 70 degrees C, similar to 75 degrees C, and similar to 80 degrees C yielded similar to 4.2 +/- 0.2 nm, similar to 4.3 +/- 0.3 nm,similar to 4.1 +/- 0.2 nm, and similar to 4.7 +/- 0.5 nm, respectively. This, along with Delta G (plateaued values) and translocation time profiles, points to the possibility of flagellin misfolding at similar to 80 degrees C.