title: Effect of pH and ionic strength on the cytolytic toxin Cyt1A: a fluorescence spectroscopy study.
abstract: Cyt1A is a cytolytic toxin produced by Bacillus thuringiensis var. israelensis. Due to its toxicity in vivo against mosquitoes and black flies, it is used as an environmentally friendly insecticide, although its mode of action is not completely understood. The toxin is membrane-active, but its membrane-bound conformation is unknown. In the absence of direct structural data, fluorescence spectroscopy was used to obtain indirect information on Cyt1A conformation changes in the environment mimicking the vicinity of the lipid membrane (lower pH and increased ionic strength). With decreasing pH, Cyt1A's surface hydrophobicity increased, which is consistent with an increased interaction with model membranes at low pH values, as observed previously. The pK(a) value of this conformation change is 4.4+/-0.1. Intrinsic tryptophan fluorescence decreased with decreasing pH, and the pK(a) value was the same as the one determined with synthetic probes. The protein has two types of hydrophobic binding sites, and at low pH these sites bind more probe molecules (bis-ANS) with a higher affinity than at pH 7.4. When bound to the lipid, the toxin exhibited conformation similar to the molten-globule state and showed some characteristics also observed at low pH. However, the conformation of the lipid-bound toxin did not depend on pH. Neutral salts like NaCl and KCl induced conformational changes at neutral pH, but not at low pH. These changes were most probably due to specific interactions of the salt ions with the charged amino acids on the protein surface rather than due to general effects such as Hofmeister and Debye-Hückel. Our results might contribute to elucidating the mode of action of Cyt1A, and perhaps also to improving the formulation of the insecticidal preparations.
authors.affiliation: Department of Chemistry and Biochemistry, PEC 436, University of Southern Mississippi, Hattiesburg, MS 39406-5043, USA.
authors.forename: Slobodanka D
authors.initials: SD
authors.lastname: Manceva
authors.forename: Marianne
authors.initials: M
authors.lastname: Pusztai-Carey
authors.forename: Peter
authors.initials: P
authors.lastname: Butko
mesh_terms: D001413:Bacillus thuringiensis / Q000737:chemistry; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D001665:Binding Sites; D004731:Endotoxins / Q000737:chemistry*; D006460:Hemolysin Proteins; D006863:Hydrogen-Ion Concentration; D007306:Insecticides / Q000737:chemistry*; D007700:Kinetics; D009994:Osmolar Concentration; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014364:Tryptophan