Interactions between the synaptic protein α-Synuclein and cellular membranes may be relevant both to its native function as well as its role in Parkinson's disease. We use single molecule Förster resonance energy transfer to probe the structure of α-Synuclein bound to detergent micelles and lipid vesicles. We find evidence that it forms a bent-helix when bound to highly curved detergent micelles, whereas it binds more physiological 100 nm diameter lipid vesicles as an elongated helix. Our results highlight the influence of membrane curvature in determining α-Synuclein conformation, which may be important for both its normal and disease-associated functions.α-Synuclein (AS) is the primary protein constituent of cytoplasmic Lewy bodies and Lewy neurites that are the pathological hallmark of Parkinson's Disease (PD)(1 , 2). Although it is strongly implicated in disease progression (3) the precise role of AS in PD is unclear. The native function of AS is also poorly understood, although evidence suggests that it may play a role both in maintaining neuronal plasticity and in the regulation of synaptic vesicle recycling (4 , 5).AS is disordered in solution(6) but undergoes a conformational change to an α-helical structure upon association with negatively charged membranes (7 , 8). A number of in vitro studies have characterized the interactions of AS both with detergent micelles and lipid membranes (reviewed in (9)). However, there is conflicting evidence as to whether AS demonstrates preferential affinities for specific phospholipids(7 -12) as well as if association with lipids inhibits or promotes AS aggregation or oligomerization (12 -16). A further matter of debate is the configuration of micelle or vesicle bound AS, with contrasting models proposing either an extended, continuous helix (17 -20) or two anti-parallel, non-interacting helices(21 -24), with an unstructured loop region between residues ~40-45 (Figure 1). Characterizing these conformations is of great interest, as membrane-bound structures may be pertinent both to native and disease-associated functions.Here we use single molecule Förster resonance energy transfer (FRET) to probe the helical structure of AS bound to SDS micelles and large unilamellar vesicles (LUVs). In FRET, the energy transfer efficiency (ET eff ) is dependent upon the distance between donor and acceptor fluorophores to the sixth power (25). In single molecule studies of protein conformations, each protein is labeled with a donor and an acceptor fluorophore. Photon bursts from the labeled † This work was supported by a grant from the Ellison Medical Foundation.. *To whom correspondence should be addressed: 266 Whitney Avenue, Box 208114, New Haven, CT 06520-8114; Telephone: 203-432-5342; Fax: 203-432-5175 proteins are collected as they diffuse through a diffraction-limited excitation volume. ET eff is calculated as: ET eff = I a°/ (I a°+ γI d°) , where I a° and I d° are the photon counts on the acceptor and donor channels, respectively, corrected for background and signal b...

Intrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson's and Alzheimer's diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense.

The aggregation of the protein a-synuclein (AS) is critical to the pathogenesis of Parkinson's disease. Although generally described as an unstructured monomer, recent evidence suggests that the native form of AS may be an a-helical tetramer which resists aggregation. Here, we show that N-terminal acetylation in combination with a mild purification protocol results in an oligomeric form of AS with partial a-helical structure. N-terminal acetylation of AS could have important implications for both the native and pathological structures and functions of AS. Through our demonstration of a recombinant expression system, our results represent an important step toward biochemical and biophysical characterization of this potentially important form of AS.

Both oxidative stress and aggregation of the protein α-synuclein (aS) have been implicated as key factors in the etiology of Parkinson’s disease. Specifically, oxidative modifications to aS disrupt its binding to lipid membranes, an interaction considered critical to its native function. Here we seek to provide a mechanistic explanation for this phenomenon by investigating the effects of oxidative nitration of tyrosine residues on the structure of aS and its interaction with lipid membranes. Membrane binding is mediated by the first ~95 residues of aS. We find that nitration of the single tyrosine (Y39) in this domain disrupts binding due to electrostatic repulsion. Moreover, we observe that nitration of the three tyrosines (Y125/133/136) in the C-terminal domain is equally effective in perturbing binding, an intriguing result given that the C-terminus is not thought to interact directly with membranes. Our investigations show that tyrosine nitration results in a change of the conformational states populated by aS in solution, with the most prominent changes occurring in the C-terminal region. These results lead us to suggest that nitration of Y125/133/136 reduces the membrane binding affinity of aS through allosteric coupling by altering the ensemble of conformational states and depopulating those capable of membrane binding. While allostery is a well-established concept for structured proteins, it has only recently been discussed in the context of disordered proteins. We propose that allosteric regulation through modification of specific residues in, or ligand binding to, the C-terminus may even be a general mechanism for modulating aS function.

α-Synuclein (αS) is an intrinsically disordered protein whose aggregation into ordered, fibrillar structures underlies the pathogenesis of Parkinson's disease. A full understanding of the factors that cause its conversion from soluble protein to insoluble aggregate requires characterization of the conformations of the monomer protein under conditions that favor aggregation. Here we use single molecule Förster resonance energy transfer to probe the structure of several aggregation-prone states of αS. Both low pH and charged molecules have been shown to accelerate the aggregation of αS and induce conformational changes in the protein. We find that at low pH, the C-terminus of αS undergoes substantial collapse, with minimal effect on the N-terminus and central region. The proximity of the N- and C-termini and the global dimensions of the protein are relatively unaffected by the C-terminal collapse. Moreover, although compact at low pH, with restricted chain motion, the structure of the C-terminus appears to be random. Low pH has a dramatically different effect on αS structure than the molecular aggregation inducers spermine and heparin. Binding of these molecules gives rise to only minor conformational changes in αS, suggesting that their mechanism of aggregation enhancement is fundamentally different from that of low pH.