Interplay of α-synuclein binding and conformational switching probed by single-molecule fluorescence

Abstract

We studied the coupled binding and folding of ␣-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of ␣-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple ␣-helical structures. ␣-Synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive ␣-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preencoded by the ␣-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of ␣-synuclein.A lpha-synuclein, a highly acidic 140-residue protein expressed at high levels in the human brain and enriched in presynaptic nerve termini, is a member of the growing class of intrinsically disordered proteins that adopt ordered structure upon interaction with cellular partners (1-5). This natively unfolded protein plays crucial roles in the pathogenesis of several neurodegenerative disorders including Parkinson's disease and Alzheimer's disease (6-9). Although several physiological functions have been proposed for the protein, including roles in the regulation of distinct pools of presynaptic vesicles (10, 11), maintenance of SNARE protein complexes (12), modulation of neural plasticity (13), control of dopamine neurotransmission (14), and ER-Golgi trafficking (15), its precise biological role remains unclear. Nevertheless, membrane interaction is generally believed to be a key modulator of ␣-synuclein function (16,17).Sequence analysis predicts ␣-synuclein interaction with lipid membranes through amphipathic ␣-helices encoded by 7 imperfect 11-residue repeats, approximately 4 of which are located in the highly basic N-terminal region of the protein, and 3 in the highly acidic and hydrophobic NAC region (non-A␤ component of Alzheimer's disease amyloid) (13, 18). Not surprisingly, the protein undergoes structural transitions upon binding to either brain-derived or synthetic acidic phospholipid vesicles, adopting ␣-helical conformations in the membrane-bound form (17)(18)(19). Similarly, ␣-synuclein assumes helical structu...

19F NMR studies of α‐synuclein‐membrane interactions

“…2(B)] are consistent both with this conclusion and with conclusions based on 15 N NMR data, which show that the N-terminal region of the protein binds SDS while the C-terminal region remains disordered. 13,20,24,28,31,32 Solution NMR is a powerful tool for accessing processes that occur over a range of timescales. 26 In 250 mM NaCl, SDS forms larger rod-like micelles, decreasing the tumbling rate of the a-synuclein-micelle complex.…”

“…In the a-synuclein-membrane interaction model, [21][22][23][24][25]31 $100 N-terminal residues lie on the membrane surface as an extended helix, and the remaining residues are disordered. Accordingly, the buried acyl chain should have little effect on a-synuclein interactions.…”

“…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.…”

N‐terminal acetylation is critical for forming α‐helical oligomer of α‐synuclein

“…The binding of individual detergent molecules is capable of generating a-helical structure in AS. 18 This, in combination with the properties conferred by N-terminal acetylation, may also favor oligomerization of AS. Ongoing work in our lab is aimed at clarifying this issue.…”

Ultrafast cooling reveals microsecond-scale biomolecular dynamics

“…The fluorescence of the donor dyes attached to the DNA hairpins and respective controls (TMR for HP1 and Alexa488 for HP2) as a function of time during T-jumps and iT-jumps in the microfluidic device has been measured on a confocal setup (described in detail elsewhere 25 ) with the Olympus 40X/0.90 objective lens. The observation channels in these experiments have a depth of 0.7 mm.…”