(A) Purification of α-Syn expressed in bacteria. Purified α-Syn wildtype and the three Parkinson's disease (PD)-related mutants A30P, E46K, and A53T (all tag-free constructs) were analyzed by SDS-PAGE and Coomassie staining. (B) Lipid binding of recombinant α-Syn wildtype, and of the PD mutants A30P, E46K, and A53T. α-Syn was incubated with negatively charged liposomes (30% phosphatidylserine, 70% phosphatidylcholine, or 100% phosphatidylcholine as control), 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-Syn. Top two fractions were defined as lipid-bound, and quantitated as percent of total α-Syn. Data shown are means ± SEM; n = 3. (C) Impaired lipid-binding of α-Syn decreases v-vesicle clustering. In order to measure vesicle clustering, the number of DiI-labeled v-vesicles that were docked to immobilized v-vesicles was counted, as illustrated in Figure 3. (D) Impaired lipid-binding of α-Syn increases the number of v-/t-vesicle associations. The number of fluorescent spots arising from labeled v-vesicles were counted that were docked to t-vesicles (immobilized to the imaging-surface), as illustrated in Figure 2, but in the presence of the specified α-Syn mutants without complexin-1. In all panels, error bars are standard deviations from 10 random imaging locations in the same sample channel, and *** indicates p<0.001 by the Student's t-test.