Alpha-synuclein (alphaS) is a soluble synaptic protein that is the major proteinaceous component of insoluble fibrillar Lewy body deposits that are the hallmark of Parkinson's disease. The interaction of alphaS with synaptic vesicles is thought to be critical both to its normal function as well as to its pathological role in Parkinson's disease. We demonstrate the use of fluorescence correlation spectroscopy as a tool for rapid and quantitative analysis of the binding of alphaS to large unilamellar vesicles of various lipid compositions. We find that alphaS binds preferentially to vesicles containing acidic lipids, and that this interaction can be blocked by increasing the concentration of NaCl in solution. Negative charge is not the only factor determining binding, as we clearly observe binding to vesicles composed entirely of zwitterionic lipids. Additionally, we find enhanced binding to lipids with less bulky headgroups. Quantification of the protein-to-lipid ratio required for binding to different lipid compositions, combined with other data in the literature, yields an upper bound estimate for the number of lipid molecules required to bind each individual molecule of alphaS. Our results demonstrate that fluorescence correlation spectroscopy provides a powerful tool for the quantitative characterization of alphaS-lipid interactions.

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...

The protein ␣-Synuclein (aS) is a synaptic vesicle-associated regulator of synaptic strength and dopamine homeostasis with a pathological role in Parkinson's disease. The normal function of aS depends on a membrane-associated conformation that is adopted upon binding to negatively charged lipid surfaces. Previously we found that the membrane-binding domain of aS is helical and suggested that it may exhibit an unusual structural periodicity. Here we present a study of the periodicity, topology, and dynamics of detergent micelle-bound aS using paramagnetic spin labels embedded in the micelle or attached to the protein. We show that the helical region of aS completes three full turns every 11 residues, demonstrating the proposed 11/3 periodicity. We also find that the membrane-binding domain is partially buried in the micelle surface and bends toward the hydrophobic interior, but does not traverse the micelle. Deeper submersion of certain regions within the micelle, including the unique lysine-free sixth 11-residue repeat, is observed and may be functionally important. There are no long-range tertiary contacts within this domain, indicating a highly extended configuration. The backbone dynamics of the micelle-bound region are relatively uniform with a slight decrease in flexibility observed toward the C-terminal end. These results clarify the topological features of aS bound to membrane-mimicking detergent micelles, with implications for aS function and pathology.Keywords: Synuclein; Parkinson's; amyloid; protein aggregation; membrane-associated proteins; helix periodicity ␣-Synuclein (aS) is a highly conserved presynaptic protein that plays a role in synaptic strength maintenance and dopamine homeostasis. Evidence that aS controls synaptic strength comes from neuronal cell line and knockout mouse models (Abeliovich et al. 2000;Murphy et al. 2000;Cabin et al. 2002;Schluter et al. 2003) that display impaired synaptic response to repetitive stimuli and alterations in the number of reserve pool vesicles, suggesting that aS regulates reserve synaptic vesicles called upon when readily releasable vesicles are exhausted. aS may accomplish this in part by interacting with and regulating phospholipase D (PLD) (Jenco et al. 1998;Ahn et al. 2002;Outeiro and Lindquist 2003;Payton et al. 2004), an enzyme with a purported role in vesicular trafficking (Liscovitch et al. 2000). aS also appears to be involved in regulating intracellular dopamine levels at several points of control. Expression of aS alters synaptic membrane permeability to dopamine by interacting with the human dopamine transporter (hDAT), resulting in withdrawal of hDAT from the external membrane . aS can also block dopamine synthesis by inhibiting tyrosine hydroxylase (Perez et al. 2002). In addition, aS can influence the activity of the vesicular dopamine transporter VMAT2 (Lotharius et al. 2002). Article published online ahead of print. Article and publication date are at

The expression of a small sterol transport protein, STARD4, is regulated by cholesterol levels. We show that the abundance of STARD4 regulates the sensitivity of the SREBP-2 system to changes in cholesterol, providing an additional layer of regulation in the cholesterol homeostatic mechanism.

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, β-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v∕v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of αS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly α-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type αS and the C-terminal truncation mutant αS1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type αS, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that αS is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced αS aggregation both in vitro and in vivo.is one of a number of synucleopathies in which aggregation of the protein α-Synuclein (αS) is linked to pathogenesis (1). αS is intrinsically disordered, but in the presence of lipid or detergent vesicles or micelles, adopts a highly helical structure in which its N-terminal region is membrane-bound and the C-terminal tail remains predominantly free and unstructured (2, 3). Although most PD cases are sporadic or idiopathic, three point mutations of α-Synuclein-A53T, A30P, and E46K-are associated with familial and early-onset disease (see Review (4)).In addition to its free and membrane-bound states, αS adopts partially structured intermediate conformations under low-pH or high-temperature conditions (5). A folding intermediate has also been detected at low [TFE] (6). Conditions favoring the formation of these intermediates also promote amyloid fibril growth, possibly implicating intermediate conformers as key species in the aggregation pathways.Here, we examine TFE-induced monomer conformational changes, oligomerization, and fibrillization in detail for wild-type (WT) αS, C terminally truncated WT αS (αS102), and the PDassociated αS mutants A30P, A53T, and E46K, expanding upon previous studies by Munishkina, et. al. (6) and Li, et. al. (7). This research also complements our previous fluorescence correlatio...