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

alpha-Synuclein (alpha-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that alpha-syn penetrates similar to 9-14 angstrom 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 alpha-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 alpha-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal alpha-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 angstrom) for W4 insertion.