Quantification of alpha-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy.
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.
School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853-2501, USA.
Elizabeth
E
Rhoades
Trudy F
TF
Ramlall
Watt W
WW
Webb
David
D
Eliezer
D001665:Binding Sites; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013223:Statistics as Topic; D051844:alpha-Synuclein / Q000737:chemistry*
D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D010743:Phospholipids; D051844:alpha-Synuclein