Membrane-bound alpha-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 (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 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 alphaS 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 interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS 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.

Figure 1

Top - Schematic illustrating the positions, within the lipid-binding domain of αS, of the spin-labeled sites used for distance measurements. Intra (inter) helix distances are shown in red (black) lines. Bottom - DEER signals for aS mutant Q24C/E61C in (A) POPC:POPA liposomes, (B) rod-like SDS micelles and bicelles.

Figure 2

Schematic model for the conformation of αS bound to the lipid bilayer of a bicelle based on ESR distance measurements.