Aromatic residues of Caveolin-1 binding motif of α-hemolysin are essential for membrane penetration

We have created single cysteine Caveolin-1 binding motif mutants (SCCBMMs) of staphylococcal α-HL for understanding assembly and penetration. All SCCBMMs have normal folding like α-HL as examined by limited proteolysis, intrinsic fluorescence emission, no hemolytic activity and do not form hetero oligomers with α-HL indicating that the conformational changes occurred at the cell membrane are different to that of α-HL. While modification of SCCBMMs with a membrane impermeant reagent has resulted in reduced binding, badan modification has resulted in the enhancement of badan fluorescence with time of assembly (incubation time) indicating the change in environment of the badan and the need for the penetration of the aromatic amino acids. Our studies indicate that the conformational changes are probably initiated at the Caveolin-1 binding motif and provide a basis for differential mode of interaction of the Caveolin-1 binding motif depending upon the nature of the target cell membrane.