Assessing N-Terminal Modifications On Alpha-Synuclein Structure And Function

Alpha-synuclein (aS) is a small, disordered neuronal protein whose aggregation and deposition as insoluble plaques is one of the primary pathologies associated with Parkinsonu0027s disease. Although the native function of aS remains ambiguous, it is clear that binding to lipid membranes plays an important role, with the N-terminal 100 residues forming an amphipathic helix upon binding with high affinity to anionic lipids. N-terminal acetylation is a ubiquitious post-translational modification of aS (ac-aS), although one that has been neglected in the majority of biophysical studies of aS. Previously, we showed that co-expression of aS with yeast N-terminal acetyltransferase B (NatB) complex results in ac-aS and that this modification can impact the properties of the protein. Here, the impact of N-terminal acetylation on the proteinu0027s structure and membrane binding properties is investigated using a host of biophysical techniques: NMR, light scattering, and smFRET. To disentangle the effect of N-terminal modification we examine the unmodified protein (un-aS) and ac-aS, as well as an acetylation mimic where the N-terminal methionine is replaced with asparagine (N1-aS). Chemical shift analysis suggests that both N-terminal modifications increase helicity of the first 8 residues of aS in solution, with N1-aS showing intermediate helicity, i.e. un-aS u003c N1-aS u003c ac-aS. To examine the structural changes that take place upon binding, we performed smFRET for various double-labeled constructs that span the length of the protein. We find that there are significant differences in the topology of aS bound to different detergents that propagate into the C-terminal and which depend on the type of N-terminal modification. These results may help to elucidate the mechanisms by which the protein adopts various helical structures, and how these structures depend on the presence of certain lipids.