The effects of α-synuclein on phospholipid vesicle integrity: a study using ³¹P NMR and electron microscopy

Figures & data

Figure 1.  An assay using ³¹P MAS NMR to investigate the effects of asyn on the permeability of phospholipid vesicles. The spectrum of SUVs of DMPC/DOPG at a 2:1 molar ratio shows peaks from the two lipid components on the inside (PC_i, PG_i) and outside (PC_o, PG_o) of the vesicles (top). Addition of Mn^2 + to the vesicles selectively broadens the peaks from the exposed lipids on the vesicle exterior leaving visible only the peaks from the interior lipids (middle). Subsequent addition of Triton-X100 breaks open the vesicles, which exposes both leaflets of the bilayer to Mn^2 + and no signal is observed (bottom).

Figure 2.  Results of ³¹P MAS NMR experiments to investigate the effects of asyn on the permeability of phospholipid vesicles. Spectra of initially intact SUVs composed of DOPC/DOPG, DMPC/DOPG, DOPC, DMPC and LUVs of DMPC/DOPG, after addition of Mn^2 + are shown before (left) and after (right) the addition of asyn to a lipid/protein molar ratio of 1 000:1 (a). The signal observed after the addition of protein is a measure of the amount of intact vesicles remaining. Figure b, summarizes the ³¹P signal intensity remaining after the addition of Mn^2 + and asyn to lipid/protein molar ratios 2 000:1, 1 000:1, 900:1 and 800:1 of various phospholipid compositions as shown. Intensity values are scaled with lipid alone equal to 1. All experiments were performed at 30°C.

Figure 3.  Investigation of the association of asyn peptide fragments with phospholipid vesicles. Spectra of initially intact vesicles after addition of Mn^2 + are shown before (left) and after (right) the addition of asyn(10–48), asyn(71–82) or asyn(120–140) at a lipid/peptide molar ratio of 25:1. As above, the signal observed after the addition of peptide is a measure of the amount of intact vesicles remaining.

Figure 4.  EM images of mixed phospholipid vesicles alone (a), upon addition of Mn^2 + (b), in the presence of asyn and Mn^2 + (c), and in the presence of asyn with 1 mM EDTA (d). Lipid/protein molar ratios; 1000:1. Scale bars 200 nm.

Figure 5.  EM images of zwitterionic phospholipid vesicles alone (a), upon addition of Mn^2 + (b), in the presence of asyn and Mn^2 + (c), and in the presence of asyn with 1 mM EDTA (d). Lipid/protein molar ratios; 1 000:1. Scale bars 200 nm.