Figure 6
Drp1 VD is necessary and sufficient for specific CL interactions. (A) Representative emission spectra for BODIPY-FL (donor)-labeled 6X His-tagged Drp1 VD (27.5 μM final) in the absence and presence of RhPE (acceptor; 1 mol%) in 50 mol% CL-containing liposomes (2.5 mM total lipid final). FRET was determined by the decrease in donor emission intensity at 510 nm concomitant with the FRET-sensitized increase in acceptor emission intensity at 590 nm upon donor excitation at 470 nm. The spectra were corrected for background as well as for the direct excitation of the acceptor at the donor excitation wavelength. (B) FRET efficiency, E, was determined as described under Methods for BODIPY-FL-labeled Drp1 VD (27.5 μM final) as a function of increasing CL concentration in RhPE-labeled liposomes (2.5 mM total lipid final). (C) FRET-sensitized increase in RhPE (acceptor) emission intensity is plotted as a function of increasing BODIPY-FL (donor)-labeled 6X His-tagged Drp1 VD concentration. F0 is the initial intensity of RhPE at 590 nm in the absence of donor, and F is the final intensity of RhPE at 590 nm upon incubation with donor for 30 min at room temperature. Representative FRET data are shown. (D) ITC equilibrium binding isotherm for Drp1 WT (2 μM) titrated with 50 mol% CL-containing liposomes (left), or with control 100% DOPC liposomes (right), at 25 °C. Each downward spike represents a single injection of liposomes into the sample cell. The binding constant KD in the case of 50 mol% CL-containing liposomes was determined by fitting the area under the spikes to a binding isotherm.
