Biophysics of anthrax toxin inhibition by derivatives of beta-cyclodextrin

By Ekaterina Nestorovich, NIH

Mar 17, 2009

CNLS Conference room.

Single heptameric channels of the Bacillus anthracis protective antigen, PA63, were reconstituted into planar lipid membranes and their complex kinetic behavior, open channel noise, and inhibition by cationic derivatives of cyclodextrin were analyzed. Guided by the sevenfold symmetry and predominately negative charge of the PA63 pore, we use small cyclic molecule of sevenfold symmetry, β-cyclodextrin chemically modified to add seven positive charges as an effective pore blocker. The protective action of this compound has been demonstrated on both single-molecule and whole-organism levels. By channel reconstitution and high-resolution conductance recording, we demonstrate that cationic β-cyclodextrin (AmPrβCD) interacts strongly with PA63 pore lumen, blocking PA63-induced transport at subnanomolar concentrations (in 0.1 M KCl). We show that high negative voltages from the side opposite to AmPrβCD addition drag the positively charges AmPrβCD into the channel. While PA63 itself generates a 1/f-like noise, spectral analysis of AmPrβCD-induced single channel blockages from the cis-side demonstrates good fit by a single Lorentzian power spectrum. Therefore, the AmPrβCD binding can be approximated by a two-state Markov process. We did not find any evidence for the AmPrβCD translocation through the channel. The blocker efficiently inhibits the pore when added from either cis or trans side of the membrane, however at trans-addition binding is rather irreversible within the limits of the time period of our experiment.

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