Fig. 3.
Measurement of leakage at time points after exposure to protein. Liposomes were prepared with encapsulated 70-kDa fluorescein dextran or membrane-associated Oregon Green DHPE. (A) Leakage profiles are observed by addition of the fluorescence quencher DPX at the indicated (τDPX) time points after addition of 8 μM IAPP. Dashed line shows predicted behavior at τDPX = 4,000 s for a model in which IAPP rapidly creates semipermeable pores (see main text). (B) Representative leakage profiles observed by addition of the fluorescence quencher, DPX, at the indicated (τDPX) time points after addition of 7 μM IAPP. (Inset) Statistical assessment (N = 3) of kinetics represented by (B) fitting single exponentials. Result is a plot of rate constants versus τDPX with solid line showing a single exponential fit to this data. (C) Acceleration of leakage rate upon secondary addition of protein. Liposomes were preincubated for 48 h with 7 μM IAPP. Leakage was measured via addition of DPX with (green) or without (violet) the further addition of 2 μM IAPP 30 s prior to measurement. (Inset) Statistics (N = 4) of the change in apparent leakage rate constant at time points, τDPX, after secondary addition of 2 μM protein. Starred point indicates the leakage rate constant prior to secondary protein addition. Dashed line shows the midpoint for initial equilibration of 7 μM IAPP (as determined in B). Note, rates observed in this figure are slightly elevated compared to Fig. 1 as a result of protocol-specific sample manipulation effects on protein concentration (see SI Materials and Methods).