**7.4 Analysis of α-synuclein structure with other methods**

Contradictory to these current models of membrane-bound α-synuclein that have been deduced mostly from NMR studies, limited proteolysis experiments have indicated that the C-terminal part of membrane-bound α-synuclein has a more rigid structure. The negatively charged C-terminus appears to bind Ca2+ in the presence of SDS micelles, and in doing so it becomes sufficiently rigid and structured to resist extensive proteolysis (de Laureto et al., 2006). In another study based on site-directed fluorescence labeling, they also examined the effects of Ca2+ on the acidic tail conformation of lipid-bound α-synuclein (Tamamizu-Kato et al., 2006). Here, they suggested that the Ca2+ either bridges α-synuclein to the membrane, possibly by coordinating with the negative charge on the α-synuclein acidic tail and the acidic head-groups in the phospholipid bilayer, or it facilitates the traversing of the membrane bilayer by this segment of α-synuclein (Tamamizu-Kato et al., 2006).

Another study highlighted the role of the physical parameters of the membrane mimetic in determining the α-synuclein conformation (Trexler & Rhoades, 2009). Single molecule Förster resonance energy transfer was used to probe the helical structure of α-synuclein bound to SDS micelles and LUVs. Single and double Cys α-synuclein mutants were engineered to allow for site-specific labeling with maleimide fluorophores. When bound to highly curved detergent micelles, α-synuclein formed a bent-helix, whereas the structure of the elongated helix was adopted when bound to the more physiological 100-nm-diameter lipid vesicles (Trexler & Rhoades, 2009).

Single-molecule Förster resonance energy transfer was also used to provide evidence for the structural interplay between the broken and extended α-helix structures of α-synuclein, as induced by the binding of α-synuclein to SDS and phospholipid SUVs (Ferreon et al., 2009). The switch between a broken and an extended helical structure can be triggered by changing the concentrations of the binding partners or by varying the curvature of the binding surfaces presented by the micelles or bilayers composed of SDS. The use of lipid vesicles of various compositions showed that a low fraction of the negatively charged lipids, as similar to that found in biological membranes, was sufficient to drive α-synuclein binding and folding that resulted in the induction of the extended helical structure (Ferreon et al., 2009).

The structure of the N-terminal domain of α-synuclein has also been determined using theoretical methods (Mihajlovic & Lazaridis, 2008). This computional study of the binding of truncated α-synuclein (residues 1-95) to planar bilayers showed that α-synuclein forms a bent helix, with the largest bend around residue 47. This bending of the helix was not due to the protein sequence or membrane-protein interactions, but to the collective motions of the long helix (Mihajlovic & Lazaridis, 2008).
