Journal
PROTEIN SCIENCE
Volume 18, Issue 9, Pages 1882-1895Publisher
WILEY
DOI: 10.1002/pro.199
Keywords
prepore-to-pore conversion; protein-detergent complex; conformational changes; biophysical characterization
Categories
Funding
- NIH [Al022021, Al19807, Al057159]
- Gates Foundation
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R37AI019807, U54AI057159, R37AI022021, R01AI019807, R01AI022021] Funding Source: NIH RePORTER
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Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self-associates to form a heptameric ring-shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore-like state. N-tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion-competent 440-kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore-like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14-solubilized pore showed an extended, mushroom-shaped structure. Circular dichroism measurements revealed an increase in a helix and a decrease in beta structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2 beta 2-2 beta 3 loop transforms into the transmembrane segment of the beta-barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to a structure is dependent on residues of the luminal 2 beta 11-2 beta 12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.
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