Segmental Deuteration of α-Synuclein for Neutron Reflectometry on Tethered Bilayers

Neutron reflectometry (NR) is uniquely suited for studying protein interaction with phospholipid bilayers along the bilayer normal on an angstrom scale. However, NR on its own cannot discern specific membrane-bound regions due to a lack of scattering contrast within a protein. Here we report the successful coupling of native chemical ligation (NCL) and NR to study α-synuclein (α-syn), a membrane-binding neuronal protein central in Parkinson's disease. Two α-syn variants were generated where either the first 86 or last 54 residues are deuterated, allowing for region-specific contrast within the protein and the identification of membrane interacting residues by NR. Residues 1-86 are positioned at the hydrocarbon/headgroup interface of the outer leaflet, whereas the density distribution of the 54 C-terminal residues ranges from the hydrocarbon region to the aqueous environment. Coupling of NCL and NR should have broad utility in studies of membrane protein folding.