Effect of Ser129 pseudophosphorylation on asyn monomer binding to lipid vesicles.

<p>aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ were coincubated with POPC- and DPPC-SUV⁴⁸⁸. Schematic A. demonstrates the appearance of vesicles, protein monomers and oligomers in 2D FIDA histograms (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098906#pone.0098906-Hogen1" target="_blank">[7]</a>). B. aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ monomers show no interactions with POPC-SUV. Conversely, extensive monomer binding to DPPC-SUV is seen irrespective of pseudophosphorylation status. C. Quantitative FIDA analysis shows a mild tendency towards reduced vesicle binding of asyn129E⁶⁴⁷. D. Quantitative 2D-FIDA analysis of monomers bound to DPPC-SUV demonstrates an overall higher amount of bound monomers (total brightness), determined through brightness and concentration of bicolored particles. Furthermore, the higher fluorescence intensity of the bicolored particles (single vesicle brightness) indicates that more monomers are bound per vesicle. E. In a control experiment, asyn⁶⁴⁷ and asyn129E⁶⁴⁷ particle brightness (Q) as determined by 1D FIDA analysis (one component fit) is unchanged in presence of POPC-SUV as compared to control measurements in the absence of vesicles, confirming that no membrane binding takes place. However, Q increases in presence of DPPC-SUV. Dissolution of DPPC-SUV by SDS yields an asyn⁶⁴⁷ particle brightness similar to monomeric asyn⁶⁴⁷, indicating that oligomer formation is not induced by binding to DPPC-SUV. Levels of significance are depicted as * = p<0.05; n = 3.