FIGURE 6.

FRET kinetics. Measurement of acceptor fluorescence at 570 nm as a function of time of a mixed composed by HlyA K344C labeled with donor and acceptor plus control erythrocytes (black line) and cholesterol-depleted erythrocytes (light gray line) are shown. Measurement of a mixture of unlabeled and labeled with acceptor HlyA K344C with control erythrocytes (dark gray line) was done as FRET negative control. Assays were performed at a ratio of 5 μg of total toxin per 100 μg of phospholipids (erythrocytes membranes). The excitation monochromator was set at 480 nm, and the emission monochromator was set at 570 nm. Alexa-546 emission was measured at a rate of 25 samples/s during 240 s, at 37 °C. The curves represent the average value of three independent experiments obtained from five replicates each.