title: Interaction of the delta-endotoxin CytA from Bacillus thuringiensis var. israelensis with lipid membranes.
abstract: We investigated the binding of CytA, a cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis, to small unilamellar lipid vesicles (SUV) and the accompanying changes in the overall CytA conformation. From the titration of tryptophan fluorescence with SUV, we determined the apparent association constants of 3500 M-1 and 11000 M-1 for the protoxin CytA27 and the proteolytically activated toxin CytA24, respectively. Inclusion of a negatively charged lipid or a positively charged lipid analog in the membrane did not affect the binding parameters, which suggests that membrane binding is not driven by electrostatic interactions. A decrease in the intensity of the CytA tryptophan fluorescence upon interaction with lipids and the absence of a blue shift in remaining fluorescence indicate that the tryptophan-containing regions of the protein do not significantly penetrate into the hydrophobic core of the lipid bilayer. This finding was corroborated by the lack of additional quenching by brominated or spin-labeled lipids, irrespective of the location of the quenching moiety in the depth of the bilayer. However, the interaction with lipids decreases quenching with the soluble quenchers acrylamide and KI, and the remaining fluorescence is blue-shifted. The observed decrease in fluorescence anisotropy upon membrane binding is not consistent with simple immobilization of CytA on the surface of SUV. We showed by FTIR spectroscopy and differential scanning calorimetry (DSC) that binding to the membrane causes a significant loosening of the protein structure. This is consistent with the fluorescence quenching and anisotropy data. Our experiments provide evidence against CytA's substantially penetrating the lipid bilayer and creating well-defined proteinaceous channels.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6. pbutko@channing.harvard.edu
authors.forename: P
authors.initials: P
authors.lastname: Butko
authors.forename: F
authors.initials: F
authors.lastname: Huang
authors.forename: M
authors.initials: M
authors.lastname: Pusztai-Carey
authors.forename: W K
authors.initials: WK
authors.lastname: Surewicz
mesh_terms: D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry*; D001427:Bacterial Toxins; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004731:Endotoxins / Q000737:chemistry*; D006460:Hemolysin Proteins; D007700:Kinetics; D008081:Liposomes; D010713:Phosphatidylcholines; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D013113:Spin Labels; D013816:Thermodynamics; D014364:Tryptophan
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008081:Liposomes; D010713:Phosphatidylcholines; D013113:Spin Labels; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014364:Tryptophan