title: Recombinant anthrax protective antigen: Observation of aggregation phenomena by TEM reveals specific effects of sterols.
abstract: Negatively stained transmission electron microscope images are presented that depict the aggregation of recombinant anthrax protective antigen (rPA83 monomer and the PA63 prepore oligomer) under varying in vitro biochemical conditions. Heat treatment (50°C) of rPA83 produced clumped fibrils, but following heating the PA63 prepore formed disordered aggregates. Freeze-thaw treatment of the PA63 prepore generated linear flexuous aggregates of the heptameric oligomers. Aqueous suspensions of cholesterol microcrystals were shown to bind small rPA83 aggregates at the edges of the planar bilayers. With PA63 a more discrete binding of the prepores to the crystalline cholesterol bilayer edges occurs. Sodium deoxycholate (NaDOC) treatment of rPA83 produced quasi helical fibrillar aggregate, similar but not identical to that produced by heat treatment. Remarkably, NaDOC treatment of the PA63 prepores induced transformation into pores, with a characteristic extended ß-barrel. The PA63 pores aggregated as dimers, that aggregated further as angular chains and closed structures in higher NaDOC concentrations. The significance of the sterol interaction is discussed in relation to its likely importance for PA action in vivo.
authors.affiliation: Institute of Zoology, University of Mainz, 55099 Mainz, Germany. Electronic address: rharris@uni-mainz.de.
authors.forename: J Robin
authors.initials: JR
authors.lastname: Harris
authors.affiliation: Fujifilm Diosynth Biotechnologies, Belasis Avenue, Billingham TS23 1LH, UK.
authors.forename: Andrei
authors.initials: A
authors.lastname: Soliakov
authors.affiliation: Envigo, Wooley Road, Alcon bury, Huntingdon, Cambridgeshire PE28 4HS, UK.
authors.forename: Allan
authors.initials: A
authors.lastname: Watkinson
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne NE2 4HH, UK.
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
mesh_terms: D000942:Antigens, Bacterial / Q000187:drug effects / Q000235:genetics / Q000648:ultrastructure*; D001427:Bacterial Toxins / Q000235:genetics; D002784:Cholesterol / Q000737:chemistry / Q000494:pharmacology; D003460:Crystallization; D003840:Deoxycholic Acid / Q000008:administration & dosage / Q000494:pharmacology; D005615:Freezing; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008958:Models, Molecular; D016624:Negative Staining; D066329:Protein Aggregates / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000648:ultrastructure; D013261:Sterols / Q000494:pharmacology*
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D066329:Protein Aggregates; D011994:Recombinant Proteins; D013261:Sterols; C030325:anthrax toxin; D003840:Deoxycholic Acid; D002784:Cholesterol
keywords: Aggregation; Anthrax protective antigen; Cholesterol microcrystal; Negative staining; PA63 pore; PA63 prepore; Sodium deoxycholate; Transmission electron microscopy; rPA83