Quantification of α-Synuclein Binding to Lipid Vesicles Using Fluorescence Correlation Spectroscopy

Abstract

Alpha-synuclein (alphaS) is a soluble synaptic protein that is the major proteinaceous component of insoluble fibrillar Lewy body deposits that are the hallmark of Parkinson's disease. The interaction of alphaS with synaptic vesicles is thought to be critical both to its normal function as well as to its pathological role in Parkinson's disease. We demonstrate the use of fluorescence correlation spectroscopy as a tool for rapid and quantitative analysis of the binding of alphaS to large unilamellar vesicles of various lipid compositions. We find that alphaS binds preferentially to vesicles containing acidic lipids, and that this interaction can be blocked by increasing the concentration of NaCl in solution. Negative charge is not the only factor determining binding, as we clearly observe binding to vesicles composed entirely of zwitterionic lipids. Additionally, we find enhanced binding to lipids with less bulky headgroups. Quantification of the protein-to-lipid ratio required for binding to different lipid compositions, combined with other data in the literature, yields an upper bound estimate for the number of lipid molecules required to bind each individual molecule of alphaS. Our results demonstrate that fluorescence correlation spectroscopy provides a powerful tool for the quantitative characterization of alphaS-lipid interactions.

19F NMR studies of α‐synuclein‐membrane interactions

“…Nevertheless, the strength of a-synuclein-membrane interaction varies, even for acid phospholipids with the same charge, indicating that head group charge is not the only factor. 30 We also find that a-synuclein prefers POPA-containing SUVs to POPS-containing SUVs, which agrees with earlier reports. 15,19 This observation reinforces the idea that although electrostatic interaction plays a significant role, other types of interactions are also involved.…”

“…41 Others report that a-synuclein binds weakly to phospholipids with neutral head groups, such as PC and PE. 30,40 Our data show that a-synuclein prefers negatively charged phospholipids over neutral phospholipids. Nevertheless, the strength of a-synuclein-membrane interaction varies, even for acid phospholipids with the same charge, indicating that head group charge is not the only factor.…”

“…Negatively charged head groups were reported to have a higher affinity in some studies, 15,19,30,40 but not in others. 41 Others report that a-synuclein binds weakly to phospholipids with neutral head groups, such as PC and PE.…”

“…More specifically, 15 N intensity and relaxation data from titration of SUVs sugget there exists several binding modes in which the first 25 residues adopt a helical state that anchor the interaction with SUVs. 26,27 A variety of other techniques, including circular dichroism spectropolarimetry, 12,13,19,28 fluorescence spectroscopy, 21,[29][30][31] and EPR [21][22][23][24][25] have been used to study how the phospholipid composition of vesicles affects a-synuclein affinity. Although these studies indicate that the properties of phospholipid membranes (i.e., charge, curvature, size, and the identity of the acyl chains) affect binding, there is no consensus, and some studies are contradictory.…”

Mitochondrial translocation of α-synuclein is promoted by intracellular acidification

“…The well-known lipid binding properties of α-synuclein [51], led us to investigate whether binding at acidic pH is mediated by mitochondria-specific lipids. Mitochondrial membranes differ from other cellular membranes in that they contain a high proportion of cardiolipin (CL), about 10-20% [52].…”