Characterization of Flagellar Filaments and Flagellin through Optical Microscopy and Label-Free Nanopore Responsiveness
- Y. M. Nuwan D. Y. BandaraY. M. Nuwan D. Y. BandaraDepartment of Mechanical Engineering, Southern Methodist University, Dallas, Texas 75275, United StatesMore by Y. M. Nuwan D. Y. Bandara
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- Jiannan TangJiannan TangDepartment of Mechanical Engineering, Southern Methodist University, Dallas, Texas 75275, United StatesMore by Jiannan Tang
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- Jugal SahariaJugal SahariaDepartment of Mechanical Engineering, Southern Methodist University, Dallas, Texas 75275, United StatesMore by Jugal Saharia
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- Louis William RogowskiLouis William RogowskiDepartment of Mechanical Engineering, Southern Methodist University, Dallas, Texas 75275, United StatesMore by Louis William Rogowski
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- Chi Won AhnChi Won AhnNano-Materials Laboratory, National NanoFab Center, Daejeon 34141, Republic of KoreaMore by Chi Won Ahn
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- Min Jun Kim*Min Jun Kim*E-mail: [email protected]Department of Mechanical Engineering, Southern Methodist University, Dallas, Texas 75275, United StatesMore by Min Jun Kim
Abstract
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 ∼65 °C (most commonly reported temperature), ∼70 °C, ∼75 °C, and ∼80 °C to investigate the effect of temperature (Tdepol) on depolymerization. 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, ∼70 °C, ∼75 °C, and ∼80 °C yielded ∼4.2 ± 0.2 nm, ∼4.3 ± 0.3 nm, ∼4.1 ± 0.2 nm, and ∼4.7 ± 0.5 nm, respectively. This, along with ΔG (plateaued values) and translocation time profiles, points to the possibility of flagellin misfolding at ∼80 °C.
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This article is cited by 12 publications.
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