Despite the substantial attention paid to alpha-syn membrane binding properties, direct measurements of the penetration depth of alpha-syn within the bilayer and the role of specific N-terminal residues in mediating membrane association have not yet been reported. Moreover, while numerous studies have focused on polypeptide conformational dynamics upon alpha-syn-membrane binding, insights into how the protein interaction influences phospholipid bilayer structure remain limited. Using multiple experimental and computational methodologies, we determined the depth of bilayer penetration of full length alpha-syn as well as the participation of specific N-terminal residues. Specifically, we evaluated N-terminal alpha-syn bilayer penetration depth by measuring fluorescence changes of a single Trp mutant (F4W) upon binding to phospholipid vesicles containing bromine, a heavy-atom, collisional quencher. We found that W4 resides 6-11 angstroms from the bilayer center or at penetration depth of 9-14 angstroms. To assess the depth of alpha-syn in a bilayer from the perspective of the membrane, neutron reflectometry (NR) and a surface-stabilized sparsely tethered bilayer lipid membrane (stBLM) were employed. Neutrons are particularly well suited for the study of biological materials because, unlike electrons and x-rays, neutrons have highly disparate signal sensitivities for hydrogen and its isotope deuterium with de Broglie wavelengths on a molecular length scale (approx. 5 angstroms). Organic layers which contain different number of hydrogen atoms, such as lipid headgroups and hydrocarbons in a membrane, can be readily distinguished and characterized. One advantage unique to NR is that both membrane-bound alpha-syn as well as changes in phospholipid bilayer properties resulting from protein binding can be simultaneously monitored. NR measurements indicate that substantial alpha-syn is associating with the membrane at both the headgroup and hydrocarbon region (13.1 angstroms thick protein region penetrating 4.2 angstroms into the outer leaflet hydrocarbons), consistent with bromine quenching data. Notably, the association of alpha-syn resulted in sizable reduction (1.44 angstroms) in bilayer thickness. To our knowledge, this is the first reported result to directly quantify alpha-syn induced membrane thinning. While the absolute value of 1.44 angstroms appears to be modest, we note that this value represents an average over the whole stBLM surface area and that localized thinning could be much higher. alpha-Syn penetration into the outer leaflet may explain why the protein has a preference for highly curved membranes and can even induce membrane curvature under certain conditions. The shallow insertion could induce membrane curvature to compensate for changes to headgroup packing and for differences in the area between the inner and outer leaflets. While the completeness of the stBLM was maintained during the course of our experiments (95-100%), the measured membrane thinning could represent a first step leading to disruption of the bilayer which has been suggested as a potential pathogenic mechanism. We investigated the involvement of specific N-terminal residues in membrane penetration by using N-terminal alpha-syn polypeptides containing W4. We identified that the first fifteen residues (P15) nearly recapitulate the features of the full-length protein (i.e. partition constants, molecular mobility and insertion of the W4 sidechain into the bilayer)and found that as little as the first four N-terminal residues are sufficient for vesicle binding. Interestingly, while at least one imperfect amphipathic repeat sequence (KAKEGV) is required for alpha-helical structure, secondary structural formation has little effect on membrane affinity. To develop a molecular framework of N-terminal residue interactions with the phospholipid bilayer, all-atom molecular dynamics simulations were performed on the P15 peptide submerged into a bilayer. Simulation results are highly consistent with experimental data indicating a broad low energy region (8.5-14.5 angstroms) for W4 insertion. Our finding that isolated N-terminal peptides can bind to the membrane with comparable affinity as the full length protein could have biological implications. Natural membranes, such as synaptic vesicles, are decorated with integral and peripheral membrane proteins limiting the amount of free lipid surface. Thus, alpha-syn may not be able to bind to the membrane in a fully extended alpha-helical conformation in vivo. Rather, specific regions of alpha-syn may bind to the membrane leaving other sites available for interaction with other biomolecules.
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