Depth of α-Synuclein in a Bilayer Determined by Fluorescence, Neutron Reflectometry, and Computation

Abstract

α-Synuclein (α-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that α-syn penetrates ∼9-14 Å into the outer leaflet of the bilayer, with a substantial portion of the membrane-bound polypeptide extending into the aqueous solvent. For the first time, to our knowledge, we used NR to obtain direct quantitative evidence of α-syn-induced membrane thinning. To examine the effect of specific residues on membrane penetration depths, we used a series of W4-containing N-terminal peptides. We identified that the first 15 residues (P15) nearly recapitulate the features of the full-length protein (i.e., partition constants, molecular mobility, and insertion of the W4 side chain into the bilayer), and found that as few as the first four N-terminal residues are sufficient for vesicle binding. Although at least one imperfect amphipathic repeat sequence (KAKEGV) is required for α-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal α-syn model for bilayer interactions, we performed molecular-dynamics simulations of the P15 peptide submerged in a bilayer. The simulation results are highly consistent with experimental data indicating a broad low-energy region (8.5-14.5 Å) for W4 insertion.

Adsorption of α-Synuclein to Supported Lipid Bilayers: Positioning and Role of Electrostatics

“…A minor thinning of the bilayer to free space for the protein in the headgroup layer was needed in the modeling, which is also consistent with the previous NR study for another lipid composition and pH. 28 However, the thinning does not free enough space for the protein to be located between the outer phospholipid headgroups. A perturbed acyl chain packing with increased water content in the acyl chain layer is needed in addition to the thinning of the bilayer to observe a good fit while preserving stoichiometric relations.…”

Structural Features of Membrane-bound Glucocerebrosidase and α-Synuclein Probed by Neutron Reflectometry and Fluorescence Spectroscopy

“…5C). The observed changes cannot simply be due to crowding at the membrane surface; ␣-syn in the absence of GCase has been measured at comparable membrane coverage, and no changes have been observed resembling those seen for the complex (34). Unfortunately, we are near the limit of detection by NR, so lowering protein concentrations of ␣-syn and GCase below 300 nM is not feasible at this time; therefore, it is uncertain to what extent steric contacts might be perturbing the NR results.…”

Chemical properties of lipids strongly affect the kinetics of the membrane-induced aggregation of α-synuclein

“…In general, the amino acid sequences of these peripheral proteins are characterized by patterns of hydrophobic and polar residues such that the proteins fold into amphipathic α-helices upon binding to hydrophobic patches exposed at the membrane interface (16,17). In particular, molecular dynamics simulations and neutron reflectometry studies of deposited bilayers have shown that the amphipathic helix in α-synuclein is primarily located in the vicinity of the lipid phosphate groups and the glycerol backbone (16,(23)(24)(25).…”