1. Design of lipid-binding deficient mutants of α-synuclein.
Design of lipid-binding deficient mutants of α-synuclein. A, α-Synuclein domain structure (red, lipid-binding domain; blue, protein-interaction domain), with lipid-binding deficient mutations marked in red and 11-mer sequences highlighted with black boxes. B, SDS-PAGE analysis of purified recombinant mutant α-synuclein (5 μg/lane), stained with Coomassie Brilliant Blue. C, D, Lipid binding of wild-type and mutant α-synuclein. C, Recombinant α-synuclein was incubated with negatively charged liposomes (composition: 30% phosphatidylserine (PS) and 70% phosphatidylcholine (PC)) and subjected to a flotation assay. Eight fractions were collected from top to bottom of the flotation gradient, and equal volumes of each fraction were separated by SDS-PAGE and immunoblotted for α-synuclein. D, The top two fractions were defined as lipid bound and quantitated as percentage of total α-synuclein (means ± SEMs; **p < by Mann–Whitney U test; n = 3). E, Analysis of the effect of α-synuclein mutations on membrane-binding induced α-synuclein multimerization. Recombinant α-synuclein was incubated with negatively charged liposomes (composition: 30% PS, 70% PC) and exposed to increasing concentrations of the chemical crosslinker glutaraldehyde (concentration: 0–0.5%). Equal volumes of crosslinked proteins were analyzed by immunoblotting. Arrowheads indicate α-synuclein multimers.
Jacqueline Burré et al. J. Neurosci. 2015;35:
©2015 by Society for Neuroscience