FIGURE 4.

FRET detected association between ILY and hCD59 in prepore complexes. A cysteine was introduced at amino acid residue Asp-280 in both ILY^wt and ILY^pp and was labeled with tetramethylrhodamine (A, acceptor fluorophore). A, erythrocytes were preincubated with a FITC-conjugated (D, donor fluorophore) anti-hCD59 mAb (MEM43) and then incubated with either unlabeled ILY^un (D + U, solid line) or acceptor-labeled ILY^wt (D + A, dashed line) or ILY^pp (D + A, dotted line). FRET between donor and acceptor is observed as a decrease in the fluorescence per cell (i.e. the peak will shift to the left) of the donor when the unlabeled toxin is replaced with acceptor-labeled toxin (compare D + U and D + A). No significant change in donor fluorescence was observed when acceptor-labeled PFO^pp (D + A, dotted) or unlabeled PFO^un (D + A, solid line) was used (B). GMF, geometric mean of fluorescence. *, p < 0.005 for the geometric mean of fluorescence of cells treated with ILY^pp and ILY^wt. No significant difference in the geometric mean of fluorescence of cells treated with either PFO^pp or PFO^wt was observed.