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Interplay of
-synuclein binding and conformationalswitching probed by single-molecule fluorescence
Allan Chris M. Ferreon
1
, Yann Gambin
1
, Edward A. Lemke, and Ashok A. Deniz
2
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037Edited by Jose N. Onuchic, University of California San Diego, La Jolla, CA, and approved February 10, 2009 (received for review September 17, 2008)
We studied the coupled binding and folding of
-synuclein, anintrinsically disordered protein linked with Parkinson’s disease.Using single-molecule fluorescence resonance energy transfer andcorrelation methods, we directly probed protein membrane asso-ciation, structural distributions, and dynamics. Results revealed anintricate energy landscape on which binding of
-synuclein toamphiphilic small molecules or membrane-like partners modulatesconformational transitions between a natively unfolded state andmultiple
-helical structures.
-Synuclein conformation is not con-tinuously tunable, but instead partitions into 2 main classes offoldinglandscapestructuralminima.Theswitchbetweenabrokenandanextendedhelicalstructurecanbetriggeredbychangingtheconcentration of binding partners or by varying the curvature ofthebindingsurfacespresentedbymicellesorbilayerscomposedofthe lipid-mimetic SDS. Single-molecule experiments with lipidvesicles of various composition showed that a low fraction ofnegatively charged lipids, similar to that found in biological mem-branes, 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 preen-coded by the
-synuclein amino acid sequence, and are tunable bysmall-molecule supramolecular states and differing membraneproperties, suggesting novel control elements for biological andamyloid regulation of
-synuclein.
amyloid
misfolding
Parkinson’s disease
protein folding
supramolecular
A
lpha-synuclein, a highly acidic 140-residue protein ex-pressed at high levels in the human brain and enriched inpresynaptic nerve termini, is a member of the growing class of intrinsically disordered proteins that adopt ordered structureupon interaction with cellular partners (1–5). This nativelyunfoldedproteinplayscrucialrolesinthepathogenesisofseveralneurodegenerative disorders including Parkinson’s disease and Alzheimer’s disease (6–9). Although several physiological func-tions have been proposed for the protein, including roles in theregulation 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 roleremains unclear. Nevertheless, membrane interaction is gener-ally believed to be a key modulator of 
-synuclein function(16, 17).Sequence analysis predicts
-synuclein interaction with lipidmembranes through amphipathic
-helices encoded by 7 imper-fect 11-residue repeats, approximately 4 of which are located inthe highly basic N-terminal region of the protein, and 3 in thehighly acidic and hydrophobic NAC region (non-A
componentof Alzheimer’s disease amyloid) (13, 18). Not surprisingly, theprotein undergoes structural transitions upon binding to eitherbrain-derived or synthetic acidic phospholipid vesicles, adopting
-helical conformations in the membrane-bound form (17–19).Similarly,
-synuclein assumes helical structures upon interac-tion with micellar SDS, a well-characterized phospholipidmimicwidelyappliedin
-synucleinbiophysicalinvestigations(3,20–25).SeveralensemblebiophysicalmethodsincludingNMR(20,21,26, 27), EPR (28–30), CD (21, 22, 31, 32) and fluorescencespectroscopy (33, 34) have provided valuable insights into thestructural features of disordered and folded
-synuclein. In thecontext of membrane-binding, solution NMR studies performedin the presence of SDS spherical micelles demonstrated thatmicelle-bound
-synuclein adopts a broken
-helix structure, which consists of a pair of curved antiparallel helices connectedby a well-ordered extended linker, and followed by a shortextended region and a predominantly unstructured mobile tail(Fig. 1
 A
) (20, 21, 26). EPR analyses of 
-synuclein derivativesbound to small unilamellar lipid vesicles (SUVs) have providedevidence for an elongated helical structure devoid of significanttertiary packing (28), or suggested a broken helical structuremore recently (29, 30). To relate these reported micelle- and vesicle-boundexperimentally-observedstructures,wepreviouslycarried out a detailed ensemble thermodynamic characterizationof the SDS-induced folding of 
-synuclein in broad ranges of SDS concentration, temperature, and pH, using CD spectros-copy, providing evidence for an inherently multistate
-synuclein folding (22). However, long-range structural anddynamics information critical for the unambiguous structuralassignment of the protein thermodynamic states were unavail-able from our ensemble study.To better understand the complexities of 
-synuclein folding, we turned to single-molecule experiments, which avoid loss of information due to ensemble averaging (35–40) and thus benefitstudies of protein folding landscapes (41–43) by permittingmultiple structural distributions and coexisting populations to beresolved and examined in a more straightforward and model-independent manner. Here, we use single-molecule fluorescenceresonance energy transfer (smFRET) as a long-range distanceruler (44) to provide unequivocal evidence for the structuralinterplay between the broken and extended
-helix structuresobserved for the protein, induced by binding to SDS andphospholipid SUVs. In addition, correlation analysis of FRETfluctuations [FCS-FRET (45)] was carried out to quantify thestructural dynamics of the different
-synuclein conformations.
Results and Discussion
smFRET Maps a Multistate Landscape for SDS-Induced
-SynucleinFolding.
smFRET was used to probe the coupled folding andbinding of 
-synuclein in the presence of SDS, a negativelycharged lipid mimetic that is well-characterized, and widely-usedfor studying
-synuclein structure and function (3, 20–25). SDSprovides a simple model for biological membranes, and enablesthe precise tuning of the properties of binding partners over a
Author contributions: A.C.M.F., Y.G., and A.A.D. designed research; A.C.M.F. and Y.G.performed research; E.A.L. contributed new reagents/analytic tools; A.C.M.F., Y.G., E.A.L.,and A.A.D. analyzed data; and A.C.M.F., Y.G., E.A.L., and A.A.D. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.
1
A.C.M.F. and Y.G. contributed equally.
2
To whom correspondence should be addressed. E-mail: deniz@scripps.edu.This article contains supporting information online atwww.pnas.org/cgi/content/full/ 0809232106/DCSupplemental.
www.pnas.org
cgi
doi
10.1073
pnas.0809232106 PNAS Early Edition
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broad range of concentrations, allowing exploration of interac-tions with both small-molecule amphiphiles at low concentra-tions and membrane-like surfaces at higher concentrations. ForFRET detection of 
-synuclein conformational changes, theprotein was dual-labeled at residue positions 7 and 84 with donor(Alexa Fluor 488) and acceptor (Alexa Fluor 594) fluorescentdyes (Fig. 1
 A
), resulting in a mixture of Donor-Acceptor- and Acceptor-Donor-labeled proteins at the two positions. On thebasis of the NMR structure of SDS spherical micelle-bound
-synuclein (26), these labeling positions flank the helical re-gions of the protein, and are ideal for FRET detection of globalstructural transitions involving long-range distance changes(20–70 Å) (36), like that between the broken and extendedhelical protein states (20–22, 26, 28). The smFRET data re-ported here are in good agreement with our previous ensembledata on wild-type
-synuclein (22), showing that dye-labelingdoes not introduce significant perturbations of protein proper-ties. In the smFRET experiments, donor and acceptor fluores-cence bursts were simultaneously recorded, and individual la-beled protein molecules freely diffused through a small confocaldetection volume (seeFig. S1)(44). FRET efficiencies (E
FRET
)for individual molecules were calculated from donor andacceptor bursts, and E
FRET
histograms were constructed (
SI  Appendix
).Isothermal protein-SDS titrations monitored by smFRET were performed using SDS concentrations that span both mo-nomeric and micellar conditions. A 3-dimensional map of rawFRET histogram data collected in the presence of backgroundunlabeled protein and presented as a function of SDS concen-tration (Fig. 1
 B
) provides a model-free protein phase diagram.Simple visual examination identifies a total of 5 distinct proteinconformations, distinguished as peaks with specific E
FRET
, andresolved independent of thermodynamic models and data fitting,highlighting the strength of smFRET measurements in chartingcomplex folding landscapes (41–43). Below the SDS criticalmicelle concentration (CMC), which is
0.9 mM in the bufferconditions used in the present study (22, 24), and where the lipidmimetic is predominantly monomeric, 3 nonzero FRET peaks were detected, consistent with the natively unfolded protein (
U
conformation) specifically binding monomeric SDS molecules toform 2 differently liganded conformations (
I
and
F
). AboveCMC, where SDS is largely micellar, 2 additional nonzero FRETpeaks were detected (
I
m
and
F
m
). The designations for thedifferent conformations observed are the same as those we usedin ref. 22, and are based on the measured relative helicalcontents (
U
I
 /
I
m
F
 /
F
m
) and the type of binding partners,i.e., small molecules like SDS monomers (
I
and
F
) vs. micellesor membranes (
I
m
and
F
m
).To determine population distributions without the use of thermodynamic models (22, 46, 47), individual smFRET histo-grams were analyzed and fitted to Gaussian functions. Thecalculated fractional populations for the 5 conformations ob-served are displayed as a function of SDS concentration (Fig.1
C
). The ease with which population distributions were deter-mined without using complicated models or performing SDStitrations as a function of additional variables (e.g., temperatureor pH) demonstrates another strength of single-molecule tech-niques in revealing structural energetic information in complexsystems.In our previous thermodynamic characterization of the SDS-induced folding of 
-synuclein via ensemble far-UV CD mea-surements and protein phase diagram analyses (22), we detected3 types of protein conformational states exhibiting differentdegrees of helicity (i.e.,
U
,
I
-type and
F
-type states). Using thelong-range conformational information from the currentsmFRET measurements, we now directly observe the coexist-ence and transitions between these states, and can definitivelycorrelate the previously observed changes in helical content withstriking changes in long-range protein structure. To probe andunderstand the different protein structures in more detail, wenext analyzed the different FRET peaks and performed corre-lation experiments.
Structure and Dynamics of
-Synuclein Conformational States ProbedUsing smFRET and FCS-FRET.
Global structures of the different
-synuclein conformations detected by smFRET were charac-
Fig. 1.
-Synuclein coupled folding and binding monitored using single-molecule FRET. (
 A
) Representations of
-synuclein primary and SDS micelle-boundtertiarystructures[1XQ8;(26)],withtheN-terminal,NACandC-terminalregionscolor-codedasblue,purpleandred,respectively,andthedye-labelingpositionsindicatedbygreenandredspheres.Amixtureof2isomers(Cys8Donor-Cys84AcceptorandCys8Acceptor-Cys84Donor)fromthedual-cysteinelabelingwasusedinthesingle-moleculeexperiments—forsimplicity,only1ofthe2isomersisdepictedinthefigures.(
B
)AsummaryofthesmFRETdataontheisothermaltitrationof labeled
-synuclein (
100 pM) against SDS in the presence of 20
M WT protein. The 5 protein conformations observed are identified. (
C 
) The populationdistributions as modulated by SDS concentration, determined from nonlinear least-squares fitting of individual smFRET histograms to Gaussian functions. See
SI Appendix 
for additional details.
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doi
10.1073
pnas.0809232106 Ferreon et al.
 
terized using E
FRET
as a ruler for measuring interdye distances(see
SI Appendix
)(44). Because of the FRET distance depen-dence, protein species characterized by high E
FRET
have shortinterdye distances, and vice versa. Representative smFREThistograms acquired in solution conditions favoring a predom-inant population of 
U
,
I
,
F
or
I
m
, and
F
m
are shown in Figs. 2
 A
and 3
 A
, respectively. In addition, long-range conformationaldynamics were obtained using correlation analysis to probeFRET fluctuations on the nanosecond to millisecond timescales(
SI Appendix
)(45). FCS-FRET data for
U
,
I
,
F
and
I
m
are shownin Fig. 2
C
and summarized inFig. S2.Because a single FRET distance alone is not sufficient to make structural assignments,here we use our smFRET and FCS-FRET data in combination with our previous far-UV CD ensemble data (22), the NMRstructure of SDS spherical micelle-bound protein (26), and theelongated helical
-synuclein structure observed by EPR (28) tostructurally assign each of the observed protein conformations.The protein state
U
that exists in dilute buffer conditions isknown to be intrinsically disordered, exhibiting a ‘‘random coil’’CD spectrum (3, 22). The
U
FRET histograms obtained in 0–0.1mM SDS show a single nonzero peak, with E
FRET
of 
0.4, whichis consistent with the dimensions of an unfolded
-synuclein(interdye distance
50 Å) (Fig. 2
A
and
B
,Table S1,and
SI  Appendix
fordistancemeasurementsandlimitations).Moreover,FRET-correlation analysis revealed sub-
s fluctuations (Fig. 2
C
andFig. S2), indicative of rapid interconversion between nu- merous conformations in a disordered ensemble, consistent withthe study in ref. 27, recently observed (48) for an intrinsicallyunstructured yeast prion.Binding of SDS monomers to
U
induces formation of the
I
conformation (22), characterized by high E
FRET
(
0.8) andshort interdye distance (
34 Å) (Fig. 2
A
and
B
,Table S1).
I
displays fast (
1
s), intermediate (
1
s) and slow (
1
s)conformational fluctuations (Fig. 2
C
andFig. S2). In compari-son, the spherical micelle-bound
I
m
, shown to exhibit a broken
-helix structure (20, 21, 26), shows similarly high E
FRET
(
0.85)and short interdye distance (
37 Å) (Fig. 2
A
and
B
andTableS1). However, in contrast with
I
dynamics,
I
m
exhibits onlyintermediate-timescale conformational fluctuations (Fig. 2
C
andFig. S2). The slow ‘‘breathing’’ fluctuations of 
I
in the 5–100
s time-range correlate well with a slightly lower helicity (22)relative to I
m
.
F
, which also binds SDS monomers (22), exhibits low E
FRET
(
0.1) and long interdye distance (
67 Å) (Fig. 2
A
and
B
andTable S1). Given that all histograms have a peak at zero E
FRET
(i.e., ‘‘zero peak’’; from molecules with photobleached, missing
Fig. 2.
Structure and dynamics of
-synuclein conformations. (
 A
) smFRET histograms recorded under conditions favoring predominant population of thespecified protein species (i.e., for
U
,
I
,
F
and
I
m
conformations, 0, 0.25, 1.2, and 10 mM SDS and 20, 0, 20, and 20
M unlabeled protein were used, respectively).Thin lines represent Gaussian fits to data; bold lines are the fitted peaks for the indicated individual conformations. (
B
) Diagram representation of suggestedstructuresforthedifferentconformations.(
C 
)Correlationanalysesshowingrapid(sub-microsecond),intermediate(low-microsecond),andslow(
50
s)FRETfluctuations (yellow symbols). Green and red symbols represent pseudo cross-correlation of donor signals and cross-correlation between donor and acceptorsignals, respectively. See
SI Appendix 
for additional details.
Fig. 3.
-Synuclein folding induced by interaction with lipid membranemimics.
-SynucleininthepresenceofveryhighconcentrationofSDS(i.e.,450mM) exhibits low FRET efficiency (E
FRET
), consistent with an extended helical
F
m
structureboundtocylindricalmicelles(
 A
).Theadditionofthecosurfactanthexanol transforms spherical SDS micelles ([SDS]
50 mM) into a stack of flatbilayers, and induces a protein conformational switch from the high-E
FRET
spherical micelle-bound
I
m
form (red curve) to the low-E
FRET
bilayer-bound
F
m
species (blue curve; overlaid histogram) (
B
). Thin lines represent Gaussian fitstodata;boldlinesarethefittedpeaksfortheindicatedconformations.See
SI  Appendix 
for additional details.
Ferreon et al. PNAS Early Edition
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