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    Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance

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    Issue Date
    2013-09-17
    Author
    Naik, Subhashchandra
    Brock, Susan R.
    Akkaladevi, Narahari
    Tally, Jon
    Mcginn-Staub, Wesley
    Zhang, Na
    Gao, Philip
    Gogol, E. P.
    Pentelute, B. L.
    Collier, R. John
    Fisher, Mark T.
    Publisher
    ACS
    Type
    Article
    Article Version
    Scholarly/refereed, author accepted manuscript
    Rights
    Copyright © 2013 American Chemical Society
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    Abstract
    Domain 2 of the anthrax protective antigen (PA) prepore heptamer unfolds and refolds during endosome acidification to generate an extended 100 Å beta barrel pore that inserts into the endosomal membrane. The PA pore facilitates the pH dependent unfolding and translocation of bound toxin enzymic components, lethal factor (LF) and/or edema factor (EF), from the endosome into the cytoplasm. We constructed immobilized complexes of the prepore with the PA-binding domain of LF (LFN) to monitor the real-time prepore to pore kinetic transition using surface plasmon resonance (SPR) and bio-layer interferometry (BLI). The kinetics of this transition increased as the solution pH was decreased from pH 7.5 to pH 5.0, mirroring acidification of the endosome. Once transitioned, the LFN-PA pore complex was removed from the BLI biosensor tip and deposited onto EM grids, where the PA pore formation was confirmed by negative stain electron microscopy. When the soluble receptor domain (ANTRX2/CMG2) binds the immobilized PA prepore, the transition to the pore state was observed only after the pH was lowered to early or late endosomal pH conditions (5.5 to 5.0 respectively). Once the pore formed, the soluble receptor readily dissociated from the PA pore. Separate binding experiments with immobilized PA pores and soluble receptor indicate that the receptor has a weakened propensity to bind to the transitioned pore. This immobilized anthrax toxin platform can be used to identify or validate potential antimicrobial lead compounds capable of regulating and/or inhibiting anthrax toxin complex formation or pore transitions.
    URI
    http://hdl.handle.net/1808/23702
    DOI
    https://doi.org/10.1021/bi400705n
    Collections
    • Molecular Biosciences Scholarly Works [581]
    Citation
    Naik, S., Brock, S., Akkaladevi, N., Tally, J., Mcginn-Straub, W., Zhang, N., … Fisher, M. T. (2013). Monitoring the kinetics of the pH driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance. Biochemistry, 52(37), 6335–6347. http://doi.org/10.1021/bi400705n

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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