Bacillus thuringiensis produces cytolytic proteins (Cyt) that show toxicity against dipteran insect larvae acting directly on the cell membrane. Up to now, two different models have been proposed to explain the interaction mechanism of the cytolytic protein Cyt2Aa2 on lipid membranes: pore-forming and detergent-like action. Here we report on the interaction of Cyt2Aa2 with lipid/cholesterol bilayers at early stage (far from equilibrium) as a function of protein concentration. Quartz crystal microbalance with dissipation (QCM-D) measurements showed that the rate of protein adsorption increased with concentration, although the mass of the final protein-lipid was similar after two hours. In addition, the dissipation (compliance of the hybrid lipid/protein layer) increased with decreasing protein concentration. Furthermore, atomic force microscopy (AFM) revealed that the structure of the protein-lipid layer was concentration and time dependent. A rigid hybrid homogeneous layer was observed at protein concentrations of 50 μg/ml and 100 μg/ml after 30 min. At lower concentrations, 10 μg/ml and 17.5 μg/ml, protein adsorption on the lipid layer led to the formation of small aggregates. Interestingly, at 25 μg/ml a transition of a hole-like structure into a homogeneous layer was observed. This suggests that 25 μg/ml is a threshold concentration for the binding mechanism of Cyt2Aa2 on to lipid membranes.
Keywords: AFM; Bacillus thuringiensis; Cyt2Aa2 protein; Protein-lipid binding; QCM-D.
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