![Figure 3. The ability of α-synuclein to sense the curvature of negatively charged lipid membranes depends on its poorly developed hydrophobic face. (A) Binding of wild-type α-synuclein to small liposomes containing increasing amounts of POPS (0–80 mol%) at the expense of POPC (80–0 mol%). The remaining phospholipid was egg PC (20 mol%) and the cholesterol/phospholipid ratio was 1:2. The liposomes were obtained by extrusion through 30-nm polycarbonate filters. The plot reports the fluorescence at 530 nm as determined from the spectra shown on the left as well as from an experiment performed with sonicated liposomes of the same composition. The extruded and sonicated liposomes displayed radii in the range of 46–53 and 23–48 nm, respectively. (B) Binding of wild-type α-synuclein to size-calibrated charged liposomes (egg PC/POPS = 4:6; cholesterol/phospholipids = 1:2). The three symbols used in the right plot correspond to three independent experiments. (inset) Fluorescence (fluo) of 125 nM [NBD]α-synuclein as a function of phospholipid concentration. Color coding for sonicated and extruded liposomes is indicated on the left. Calculation of partition coefficients is described in Materials and methods. (C) Sequence of the AH region of α-synuclein highlighting the repeating character of this region, which can be divided into 11-mer repeats. The mutations harbored by the T6 mutant consist in replacing all threonine residues (pink; left) of the nonpolar region by a more hydrophobic residue (Leu or Phe, yellow; right). Position of mutated residues in the 11-mer repeats is boxed (wild type; left) or shaded (mutant; right). Color coding is as in Fig. 2; in the alignment only mutated and charged residues are colored. (D) Binding of T6 mutant α-synuclein to small liposomes containing increasing amounts of POPS at the expense of POPC as in A. (E) Binding of T6 mutant α-synuclein to size-calibrated charged liposomes as in B. Concentrations in A, B, D, and E of proteins were 125 nM and phospholipids were 150 µM. Data in A and D were fitted (dashed lines) to a sigmoidal function (y = a + bxn/(c + xn)). The dashed lines in B and E were simply used to illustrate the apparent shape of the curve for binding as a function of liposome radius.](https://cdn.rupress.org/rup/content_public/journal/jcb/194/1/10.1083_jcb.201011118/5/jcb_201011118r_rgb_fig3.jpeg?Expires=1767688277&Signature=4aXNUUlvNWr1flGCHg-wpzuEGsLOo9XK21gWdbulKyXL0Q2gdEpRTqgLJI0eDo9VPQglyMPVNvgyTsawqYUUxm9JD5MVQcUoV6oZHxWm28CEHQmHUTBXBHZa6Ru2j-xWgXBoKJQaysRFRV1cdsU71rcJgUOjZsdrzgiBFaDemR0eaHZhspDxQ1l~KuOGrZ-mpjItEMVcDdByCnh3zjqrQiyLXORvgTzrBWLGOWql0oOcz07GtKS1aMqlwuGZoikZPZCA0kgmdnOtFQ4sDCWLt8v2peXma5COipqsqsTvopoDAZwTP046fVe9xYFfWZLyKlovpnN3ljeih5NLsgW8zQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The ability of α-synuclein to sense the curvature of negatively charged lipid membranes depends on its poorly developed hydrophobic face. (A) Binding of wild-type α-synuclein to small liposomes containing increasing amounts of POPS (0–80 mol%) at the expense of POPC (80–0 mol%). The remaining phospholipid was egg PC (20 mol%) and the cholesterol/phospholipid ratio was 1:2. The liposomes were obtained by extrusion through 30-nm polycarbonate filters. The plot reports the fluorescence at 530 nm as determined from the spectra shown on the left as well as from an experiment performed with sonicated liposomes of the same composition. The extruded and sonicated liposomes displayed radii in the range of 46–53 and 23–48 nm, respectively. (B) Binding of wild-type α-synuclein to size-calibrated charged liposomes (egg PC/POPS = 4:6; cholesterol/phospholipids = 1:2). The three symbols used in the right plot correspond to three independent experiments. (inset) Fluorescence (fluo) of 125 nM [NBD]α-synuclein as a function of phospholipid concentration. Color coding for sonicated and extruded liposomes is indicated on the left. Calculation of partition coefficients is described in Materials and methods. (C) Sequence of the AH region of α-synuclein highlighting the repeating character of this region, which can be divided into 11-mer repeats. The mutations harbored by the T6 mutant consist in replacing all threonine residues (pink; left) of the nonpolar region by a more hydrophobic residue (Leu or Phe, yellow; right). Position of mutated residues in the 11-mer repeats is boxed (wild type; left) or shaded (mutant; right). Color coding is as in Fig. 2; in the alignment only mutated and charged residues are colored. (D) Binding of T6 mutant α-synuclein to small liposomes containing increasing amounts of POPS at the expense of POPC as in A. (E) Binding of T6 mutant α-synuclein to size-calibrated charged liposomes as in B. Concentrations in A, B, D, and E of proteins were 125 nM and phospholipids were 150 µM. Data in A and D were fitted (dashed lines) to a sigmoidal function (y = a + bxn/(c + xn)). The dashed lines in B and E were simply used to illustrate the apparent shape of the curve for binding as a function of liposome radius.
