Spin‐Label EPR on α‐Synuclein Reveals Differences in the Membrane Binding Affinity of the Two Antiparallel Helices

Abstract

The putative function of the Parkinson's disease-related protein alpha-Synuclein (alphaS) is thought to involve membrane binding. Therefore, the interaction of alphaS with membranes composed of zwitterionic (POPC) and anionic (POPG) lipids was investigated through the mobility of spin labels attached to the protein. Differently labelled variants of alphaS were produced, containing a spin label at positions 9, 18 (both helix 1), 69, 90 (both helix 2), and 140 (C terminus). Protein binding to POPC/POPG vesicles for all but alphaS140 resulted in two mobility components with correlation times of 0.5 and 3 ns, for POPG mole fractions >0.4. Monitoring these components as a function of the POPG mole fraction revealed that at low negative-charge densities helix 1 is more tightly bound than helix 2; this indicates a partially bound form of alphaS. Thus, the interaction of alphaS with membranes of low charge densities might be initiated at helix 1. The local binding information thus obtained gives a more differentiated picture of the affinity of alphaS to membranes. These findings contribute to our understanding of the details and structural consequences of alphaS-membrane interactions.

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation

“…If the TFE-induced F state does correspond to the highly helical membrane-bound state, then the partially helical I state, which we show is on the folding pathway to the F state, may also have a corresponding membrane-associated intermediate that could potentially be involved in membrane-induced aggregation in vivo. Indeed, evidence exists for such an intermediate; both ESR studies and recent NMR studies have reported observations of partially helical membrane-bound states of αS (30,31). Furthermore, the N terminus of αS contains a region with an elevated intrinsic helical propensity, which was proposed to nucleate helix formation upon membrane-binding by the protein (3,27).…”

19F NMR studies of α‐synuclein‐membrane interactions

“…In the a-synuclein-membrane interaction model, [21][22][23][24][25]31 $100 N-terminal residues lie on the membrane surface as an extended helix, and the remaining residues are disordered. Accordingly, the buried acyl chain should have little effect on a-synuclein interactions.…”

“…[6][7][8][13][14][15][16][17][18][19][20] Electron paramagnetic resonance (EPR) data on its complex with small unilamellar vesicles (SUVs) suggest that the first $100 residues of the monomeric protein adopt an a-helical conformation that lies on the membrane surface. [21][22][23][24][25] The last $40 residues lack defined structure and do not appear to be involved in membrane interactions. 13,21 Recent solution NMR data, however, appear incompatible with this model.…”

“…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.…”

Specificity and Kinetics of α-Synuclein Binding to Model Membranes Determined with Fluorescent Excited State Intramolecular Proton Transfer (ESIPT) Probe

“…Thus, the discrepant results and interpretations may reflect compositional issues as well as differences in key experimental parameters, such as lipid/protein ratio (47) and membrane charge (35).…”