Figure 4: Influence of mutations and buffer/lipid composition on αSyn vesicle binding.

(a) The mean residue ellipticity at 222nm of αSyn variants for increasing lipid-to-protein ratios. When not indicated otherwise, SUVs were composed of a 1:1 mixture of POPA:POPC and the buffer system was 5 mM HEPES buffer, 10 mM NaCl, pH 7.4. In addition, data for the wild-type protein in 5 mM sodium phosphate buffer, 10 mM NaCl (red) are shown. The Y39A, F94A and Y39A/F94A mutations do not attenuate αSyn's binding to liposomes (a; green, blue and cyan, respectively). (b–d) Quantification of NMR signal intensities along the αSyn sequence when bound to SUVs in the absence (red line) and presence of a 15-fold excess of PcTS (black bars). Signal intensities (I) were normalized by the values observed for αSyn in the absence of SUVs (I₀). (b) When compared with Fig. 3b, the same molar ratios of αSyn:SUVs:PcTS were used, but at an overall fivefold decreased concentration (2.5 mM SUVs, 20 μM αSyn, 300 μM PcTS). The lipid-to-protein molar ratio was 125:1. (c) NMR signal-broadening profile in the presence of vesicles composed of DOPE, DOPS and DOPC at a molar ratio of 5:3:2. Because of the lower affinity of αSyn to DOPE:DOPS:DOPC vesicles (when compared with POPA:POPC vesicles) a higher lipid-to-protein ratio was used. (d) Effect of PcTS on the liposome-induced NMR signal-broadening profile of αSyn in a 50mM sodium phosphate buffer containing 10 mM NaCl, pH 7.4. SUVs were composed of a 1:1 mixture of POPA:POPC. NMR measurements were performed at 15 °C.