title: Spectroscopic analysis of low pH and lipid-induced structural changes in type A botulinum neurotoxin relevant to membrane channel formation and translocation.
abstract: Botulinum neurotoxin (BoNT) is an extremely toxic protein to animals and humans. In its mode of action, one of its subunits mediates its translocation by integrating itself into the membrane bilayer. We have examined the membrane channel activity of type A BoNT (BoNT/A) and its heavy (H) chain in planar lipid membrane under various pH conditions to understand the possible role of the channel activity in the translocation of the BoNT/A light (L) chain under physiological conditions. Only BoNT/A H chain, and not the BoNT/A, exhibited membrane channel activity for translocation of ions. The H chain-induced increase in conductance did not require a pH gradient across the lipid membrane, although it was enhanced by a pH gradient. To understand the molecular basis of the membrane channel activity and the translocation of the L chain, the secondary structure of BoNT/A and its H and L chains were analyzed using circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy at different pH values. BoNT/A showed no structural alternation upon acidifying the buffer pH. However, an increase in beta-sheet content of BoNT/A H chain at low pH was noted when examined by FT-IR. The L chain structure significantly changed with decrease in pH, and the change was mostly reversible. In addition, the neurotoxin and its subunit chains induced a partially reversible aggregation of liposomes at low pH, which indicated their integration into the lipid bilayer. Temperature-induced denaturation studies of BoNT/A H chain indicated major structural reorganization upon its interaction with membrane, especially at low pH.
authors.affiliation: Department of Chemistry and Biochemistry, and School of Marine Science and Technology, University of Massachusetts Dartmouth, 285 Old Westport Road, Dartmouth, MA 02747, USA.
authors.forename: Fen-Ni
authors.initials: FN
authors.lastname: Fu
authors.forename: David D
authors.initials: DD
authors.lastname: Busath
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
mesh_terms: D001692:Biological Transport; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000493:pharmacokinetics*; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000096:biosynthesis*; D008564:Membrane Potentials; D010713:Phosphatidylcholines / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D013057:Spectrum Analysis
chemical_list: D007473:Ion Channels; D010713:Phosphatidylcholines; D021122:Protein Subunits; D019274:Botulinum Toxins, Type A