INTERACTION OF THE DELTA-ENDOTOXIN CYTA FROM BACILLUS-THURINGIENSIS VAR. ISRAELENSIS WITH LIPID-MEMBRANES

We investigated the binding of CytA, a cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis, to small unilamellar lipid ve sicles (SUV) and the accompanying changes in the overall CytA conforma tion. From the titration of tryptophan fluorescence with SUV, we deter mined the apparent association constants of 3500 M-1 and 11000 M-1 for the protoxin CytA27 and the proteolytically activated toxin CytA24, r espectively. Inclusion of a negatively charged lipid or a positively c harged lipid analog in the membrane did not affect the binding paramet ers, which suggests that membrane binding is not driven by electrostat ic interactions. A decrease in the intensity of the CytA tryptophan fl uorescence upon interaction with lipids and the absence of a blue shif t in remaining fluorescence indicate that the tryptophan-containing re gions of the protein do not significantly penetrate into the hydrophob ic core of the lipid bilayer. This finding was corroborated by the lac k of additional quenching by brominated or spin-labeled lipids, irresp ective of the location of the quenching moiety in the depth of the bil ayer. However, the interaction with lipids decreases quenching with th e 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 differentia l scanning calorimetry (DSC) that binding to the membrane causes a sig nificant loosening of the protein structure. This is consistent with t he fluorescence quenching and anisotropy data. Our experiments provide evidence against CytA's substantially penetrating the lipid bilayer a nd creating well-defined proteinaceous channels.