Membrane-Bound α-Synuclein Forms an Extended Helix: Long-Distance Pulsed ESR Measurements Using Vesicles, Bicelles, and Rodlike Micelles

Abstract

We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (αS) bound to small unilamellar phospholipid vesicles, rod-like SDS micelles, or lipid bicelles. By measuring distances as long as ~7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that αS does not retain a 'hairpin' structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the inter-helix linker. While the possibility of local helix discontinuities in the structure of membrane-bound αS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.Alpha-synuclein (αS) was originally discovered as a protein highly enriched in synaptosome preparations from the electric ray T. californica 1 and was later linked to both familial and sporadic Parkinson's disease (PD) through the discovery that αS point mutations or gene duplication/triplication cause familial PD and through the identification of αS as the major component of amyloid fibril aggregates present in the Lewy body deposits that are a diagnostic hallmark of PD. Both the normal function of αS and the precise relation between its aggregation and deposition in Lewy bodies and PD remain unclear. When isolated in solution, the protein is intrinsically disordered, but in the presence of lipid surfaces αS adopts a highly helical structure 2 that is believed to mediate its normal function(s). NMR-based characterization of this helical structure using detergent micelles as a membrane mimetic has shown that the protein E-mail: dae2005@med.cornell.edu; jhf@cornell.edu. adopts two extended surface-bound helices separated by a non-helical linker, that the helices are oriented in an antiparallel fashion, and that no inter-helical contacts are formed. 3-7 The slow tumbling rate of intact phospholipid vesicles precluded direct studies of the vesicle-bound conformation of αS using solution NMR methods, but it was proposed 3,8 that in the vesiclebound state, the two helices may become collinear and fuse into a single long surface-bound helix. Support for this possibility was provided by pulsed dipolar ESR (PDS) distance measurements of αS bound to different sized micelles, which showed that the helices splay further apart on the surface of larger micelles. 9 NIH Public AccessHere we use PDS, 10-13 namely 17.3 GHz DEER (cf. Su...

19F NMR studies of α‐synuclein‐membrane interactions

“…Upon binding micelles, the resonance from 3FY39 decreases and shifts from À59.9 ppm to À60.2 ppm. As shown in Figure 2(C), increasing the salt concentration to 250 mM 24 broadens the position 39 resonance beyond detection, but the resonances from the three C-terminal residues remain unchanged. Increasing the temperature to 50 C, causes the 3FY39 resonance to reappear, although it is broad [ Fig.…”

“…38 Rod-like micelles form at high salt concentrations. 24,38 Three 19 F peaks are observed from 3FY-labeled a-synuclein in buffer [ Fig. 2(A)].…”

“…Although these studies indicate that the properties of phospholipid membranes (i.e., charge, curvature, size, and the identity of the acyl chains) affect binding, there is no consensus, and some studies are contradictory. 21,22,[24][25][26][27] We show that 19 F NMR is another useful technique for assessing a-synuclein-membrane interactions. 19 F is a good reporter of conformational changes due to its sensitivity to the environment and the fact that few natural biological molecules contain fluorine.…”

“…[6][7][8][13][14][15][16][17][18][19][20] Electron paramagnetic resonance (EPR) data on its complex with small unilamellar vesicles (SUVs) suggest that the first $100 residues of the monomeric protein adopt an a-helical conformation that lies on the membrane surface. [21][22][23][24][25] The last $40 residues lack defined structure and do not appear to be involved in membrane interactions. 13,21 Recent solution NMR data, however, appear incompatible with this model.…”

“…More specifically, 15 N intensity and relaxation data from titration of SUVs sugget there exists several binding modes in which the first 25 residues adopt a helical state that anchor the interaction with SUVs. 26,27 A variety of other techniques, including circular dichroism spectropolarimetry, 12,13,19,28 fluorescence spectroscopy, 21,[29][30][31] and EPR [21][22][23][24][25] have been used to study how the phospholipid composition of vesicles affects a-synuclein affinity. Although these studies indicate that the properties of phospholipid membranes (i.e., charge, curvature, size, and the identity of the acyl chains) affect binding, there is no consensus, and some studies are contradictory.…”