Solid-State 13C NMR Reveals Annealing of Raft-Like Membranes Containing Cholesterol by the

15 Pages·2013·2.1 MB·English

by  Avigdor Leftin

13
Solid-State C NMR Reveals Annealing of Raft-Like
Membranes Containing Cholesterol by the Intrinsically
α
Disordered Protein -Synuclein
AvigdorLeftin1,ConstantinJob1,KlausBeyer1andMichaelF.Brown1,2
1-DepartmentofChemistryandBiochemistry,UniversityofArizona,Tucson,AZ85721,USA
2-DepartmentofPhysics,UniversityofArizona,Tucson,AZ85721,USA
Correspondence toMichael F.Brown: [email protected]
http://dx.doi.org/10.1016/j.jmb.2013.04.002
Editedby A.G. PalmerIII
Abstract
Misfoldingandaggregationoftheintrinsicallydisorderedproteinα-Synuclein(αS)inLewybodyplaquesare
characteristic markers of late-stage Parkinson's disease. It is well established that membrane binding is
initiatedattheN-terminusoftheproteinandaffectsbiasingofconformationalensemblesofαS.However,little
is understood about the effect of αS on the membrane lipid bilayer. One hypothesis is that intrinsically
disorderedαSaltersthestructuralpropertiesofthemembrane,therebystabilizingthebilayeragainstfusion.
Here,weusedtwo-dimensional13Cseparatedlocal-fieldNMRtostudyinteractionofthewild-typeα-Synuclein
(wt-αS) or its N-terminal (1–25) amino acid sequence (N-αS) with a cholesterol-enriched ternary membrane
system. This lipid bilayer mimics cellular raft-like domains in the brain that are proposed to be involved in
neuronal membrane fusion. The two-dimensional dipolar-recoupling pulse sequence DROSS (dipolar
recouplingon-axiswithscalingandshapepreservation)wasimplementedtomeasureisotropic 13Cchemical
shifts and 13C–1H residual dipolar couplings under magic-angle spinning. Site-specific changes in NMR
chemical shifts and segmental order parameters indicate that both wt-αS and N-αS bind to the membrane
interfaceandchangelipidpackingwithinraft-likemembranes.Mean-torquemodelingof 13C–1HNMRorder
parametersshowsthatαSinducesaremarkablethinningofthebilayer(≈6 Å),accompaniedbyanincreasein
phospholipidcross-sectionalarea(≈10 Å2).Thisperturbationischaracterizedasmembraneannealingand
entails structural remodeling of the raft-like liquid-ordered phase. We propose this process is implicated in
regulationofsynapticmembranefusionthatmaybealteredbyaggregationofαSinParkinson'sdisease.
©2013Elsevier Ltd. Allrights reserved.
Introduction connection to Parkinson's disease remain largely
unknown.4,5 One hypothesis for the function of αS is
Parkinson's disease is a debilitating neurological thatitstabilizessynapticmembranesagainstfusionin
disorderthatincreasinglyafflictstheagingpopulations the complex processes of neurotransmission that go
of industrialized countries.1 The symptoms of the awryduringParkinson'sdisease.6,7Thecurrentstudy
disease arise from neuronal cell death and are aims to further investigate the structural correlates of
associated with a drastic impairment of the dopami- thisproposal,thusaddingtoourunderstandingofαS
nergic system.2 A characteristic trait of Parkinson's interactionswiththeraft-likeneuronalmembranes.
diseaseismisfoldingandaggregationoftheproteinα- Chemicalsignalinginneuronsisregulatedbyfusion
Synuclein (αS). This intrinsically disordered protein ofsynapticvesicleswiththeactivezoneofthenerve
undergoes a series of membrane-dependent confor- terminalplasmamembrane.8TheproteinαSisknown
mational transitions3 that may be implicated in to exhibit specific interactions with the presynaptic
neurodegeneration. Ultimately, αS aggregates into membranes,9–11 including small synaptic vesicles,
fibrillar plaques—known as Lewy bodies—that accu- andthepresynapticactivezone.Ahallmarkfeatureof
mulateindopaminergicneuronswithinthesubstantia these membranes is compositional heterogeneity12
nigra.Yet,thebiologicalfunctionofαSanditsdefinitive involvingbothlipidspeciesandphasebehaviorofthe
0022-2836/$-seefrontmatter©2013ElsevierLtd.Allrightsreserved. J.Mol.Biol.(2013)425,2973–2987
2974 Annealingof Raft-Like Membranesvia α-Synuclein
bilayers. In particular, a key feature promoting the myelin (EYSM), and cholesterol (Chol) using solid-
interaction ofαS withmembranesisthe presenceof state NMR spectroscopy.21 We characterize the
detergent-insoluble microdomains (rafts)—coexis- membrane lipid behavior upon association of αS by
tence regions of liquid-disordered (l ) and liquid- two-dimensional (2D) separated local-field (SLF)
d
ordered (l ) lipid phases—that are favored by the NMR under magic-angle spinning (MAS). The SLF
o
presence of cholesterol, sphingomyelin, and unsatu- experiment DROSS (dipolar recoupling on-axis with
ratedlipids.13–15BindingofαStoraft-likemembranes scaling and shape preservation)22 permits site-
and curved single-phase bilayers is initiated by specific and simultaneous measurements of 13C
association of the N-terminal consensus sequence isotropicchemicalshiftsand 13C–1Hresidualdipolar
(N-αS)withthemembraneinterface,leadingtoacoil– couplings (RDCs) of the headgroup, backbone, and
helixconformationaltransitionoftheprotein.16Asso- acyl chains of the membrane phospholipids.21,23–28
ciationofαStosuchmembranesyieldsaninhibitionof Animportantaspectisthatisotopicenrichmentisnot
vesiclefusionthatinvolvesageneralrestructuringof required, thus providing a distinct advantage over
the lipid membrane by modulating membrane complementarysolid-state2HNMRexperiments.29,30
curvature,17soastoremovelateralphasedefectsin Usingourapproach,theisotropic 13Cchemicalshifts
compositionally or topologically heterogeneous andRDCsmonitorassociationofbothtruncatedN-αS
systems.7,16,18–20 These observations underlie our andwt-αSwiththelipidmembraneinterface.Weshow
hypothesis that αS fulfills a regulatory function in thatthisexperimentrevealslargestructuralchanges
synaptic neurotransmission by structurally remodel- in the hydrocarbon region of the membrane. The
ingneuronalraft-likemembranes(Fig.1). 13C–1H RDCs are evaluated in terms of 13C–1H
Inthisarticle,wefocusontheinteractionofwild-type segmental order parameters (S ) using a simple
CH
α-Synuclein (wt-αS) and N-αS with a canonical raft- mean-torquemodelforbilayerstructuralproperties.31
like mixture comprising 1-palmitoyl-2-oleoyl-sn-gly- Both the full-length protein and the N-terminal αS
cero-3-phosphocholine (POPC), egg yolk sphingo- peptideelicitdisorderinthephospholipidhydrocarbon
chains, resulting in thinning of the raft-like lipid
membranes.BindingofαSactsoppositelytocholes-
terol because it anneals the ordered raft-like mem-
branes.InthecontextofParkinson'sdisease,raft-like
membrane lipids may play an important role in
regulatory neurotransmitter release. The remodeling
orannealingoftheraft-likephaseobservedbysolid-
state 13C NMR addresses a molecular mechanism
suggested by previous research, whereby αS stabi-
lizes membranes against fusion. A corollary is that
misfoldingandaggregationofαSintotoxicoligomers
may lead to defective membrane remodeling, and
therefore misregulation of membrane fusion giving
risetosymptomsofParkinson'sdisease.
Results
Fig. 1. Membrane model shows biophysical mecha-
Solid-state NMR experiments probe membrane
nismforhowαSinteractswithsynapticvesiclelipids.αSis
lipids at natural isotopic abundance
apresynaptic neuronalproteinfoundinLewybodiesthat
occurinParkinson's disease.Theproposedannealingof
raft-like membrane defects by αS is depicted. (a) Raft- The 2D 13C–1H correlation experiment DROSS
based membranes constitute a heterogeneous system targets membrane components exclusively at 13C
thatisnon-ideallymixedonthenanometerscale.Clusters natural isotopic abundance. MAS allows one to
of POPC lipids (purple) or EYSM lipids (green) with investigate site-specific features and phase charac-
cholesterol (Chol; brown) coexist with mixed POPC/ teristics of complex biomembrane systems, without
EYSM/Chol regions, giving rise to local membrane de- the need of isotopic labeling as required in 2H NMR
fects.(b)NativelyunfoldedαSbindstoraft-likedefectsdue spectroscopy.32–39Thusfar,theDROSSexperiment
to sphingomyelin and cholesterol. Annealing by αS
has been implemented for the benchmark saturated
involves transient association with interfacial sites, which glycerophospholipid DMPC,22 for mixtures of the
perturbs stabilizing lipid packing interactions. Changes in
symmetric monounsaturated glycerophospholipid
thehydrophobicmembraneenvironmententailremodeling
DOPC with cholesterol,25 and for polyunsaturated
of the liquid-ordered (l ) phase of POPC and EYSM with
cholesterol, yielding aoliquid-disordered (l ) phase with a lipid species.39 To further test the performance of
d
smallerbilayerthickness(D ). DROSS on the asymmetric glycerophospholipid
B
Annealingof Raft-Like Membranesvia α-Synuclein 2975
POPC and the sphingolipid EYSM, we recorded 2D restrictedtoexperimentsconductedabovetheT of
M
spectraandextractedthe13CNMRisotropicchemical lipid membranes due to the inefficiency of the
shiftsand 13C–1HRDClineshapesforthesecompo- insensitive nuclei enhanced by polarization transfer
nents in raft-like membrane lipid mixtures. Figure 2a (INEPT) magnetization transfer in s systems. The
o
showsthestructuresandcarbonassignmentsforthe chemical shift spectra report on the nonpolar bilayer
POPC and EYSM phospholipids. The respective interior (0–45 ppm), the polar aqueous interfacial
single-component data sets obtained at 48 °C are region (50–80 ppm), and sites of unsaturation of the
showninFig.2bandcwherebothphospholipidsare acylchains(115–135 ppm).Thelargechemicalshift
inthel phase.Thistemperatureis≈10 °Cabovethe dispersionallowsuniqueassignmentstobemadefor
d
solid-ordered (s ) to l phase transition temperature the entire phospholipid molecule, which is not the
o d
T of EYSM bilayers. The DROSS experiment is case in 2H NMR spectroscopy. Large differences in
M
breadth of the RDC lineshapes for each of the
isotropic chemical shift positions are observed for
both POPC and EYSM phospholipids. Note that the
RDCsofEYSMarelargerthanthoseofPOPC,asa
consequence of greater acyl chain ordering and the
higher order–disorder transition temperature T
M
(POPC,−2 °C;EYSM,38 °C).Interactionsresponsi-
ble for the difference in the chain melting transition
temperaturesareattributedtovanderWaalscontacts
and hydrogen bonding, in accord with 2H NMR
experiments.30,40–42
Raft-like phase coexistence in ternary lipid
membranes is evident from solid-state
13C NMR spectroscopy
An equimolar mixture of the phospholipids POPC
and EYSM with cholesterol is useful as a
paradigm for raft-forming membranes. This ternary
membrane exhibits biphasic, fluid–fluid (liquid-or-
dered, l ; liquid-disordered, l ) phase coexistence
o d
over broad temperature and compositional ranges,
according to fluorescence spectroscopy and small-
angle X-ray scattering.43–46 In addition, solid-state
2H NMR has resolved the spectral signatures of
cholesterol-enriched POPC and EYSM in distinct
microenvironments.30,42
We performed the DROSS experiment to further
characterize the behavior of the raft-like membrane
lipids in aqueous dispersions prior to investigating
the αS-ternary interaction system. Figure 3a shows
the 2D SLF spectrum obtained at 48 °C for the raft-
like mixture. The 13C–1H RDC lineshape pro-
Fig. 2. SLF 13C NMR investigates raft-forming EYSM jections and isotropic 13C NMR chemical shifts
from the 2D SLF experiment are shown in Fig. 3b.
and POPC phospholipids at natural isotopic abundance.
(a)ChemicalstructuresoftheglycerophospholipidPOPC In the 13C solid-state NMR experiments, the
having palmitoyl (p) and oleoyl (o) chains, and sphingo- chemical shift interval between 29 and 32 ppm is
lipid EYSM with fatty acyl (f) and sphingosine (s) chains. associated with the (CH ) acyl chain segments.
2 n
2D dipolar-recoupled NMR spectra obtained under MAS The broad spectral region in this instance reports
at48°Careshownfor(b)POPCand(c)EYSMbilayers. on the heterogeneous raft-like microenvironments
Spectral planes are assigned to unsaturated (115– of the ternary lipid mixture. These domains are
135 ppm), headgroup plus backbone (50–80ppm), and
attributed to locally enriched pools of POPC or
acyl chain (0–40 ppm) resonances. Both phospholipids EYSM with cholesterol.14,43,47–49 To substantiate
are in the liquid-disordered (l ) phase. Site-specific
differences of 13C isotropic chemdical shifts and 13C–1H thecompositionalheterogeneityofthissystem,next
we obtained reference 13C NMR chemical shift
RDCs indicate the applicability of using the DROSS
pulse sequence to follow these spectral features in spectra for binary POPC/Chol, EYSM/Chol, and
complex raft-like ternary membranes at natural 13C POPC/EYSM membranes corresponding to the
isotopic abundance. microdomain environments. These binary mixtures
2976 Annealingof Raft-Like Membranesvia α-Synuclein
thesalientfeaturesoftheternaryspectrumrecorded
at48 °C.Here,thePOPCandEYSMpolymethylene
chemical shift regions yield significant contributions
totheternarymembranespectrum.Thisobservation
suggeststhatspectraloverlapofthe(CH ) regionis
2 n
due to chemical shift nonequivalence arising from
heterogeneous phospholipid–phospholipid and
phospholipid–cholesterol microenvironments of the
POPCandEYSMlipidsystems.
Natural abundance 13C chemical shifts indicate
selective interactions of α-Synuclein with
raft-like lipid membranes
IthasbeenreportedthatαSinteractspreferentially
atbiomembraneinterfaces,56–58 leadingtostructur-
ing of the protein due to a coil–helix transition.
Isotropic 13C chemical shifts recorded in the 2D
DROSS correlation spectra at 48 °C presented in
Fig.5ashowthatmonomericαScausesasignificant
spectral change, both within the membrane and at
themembraneinterfaceoftheternaryPOPC/EYSM/
Chol (1:1:1) system. Notably, our experiment does
not resolve 13C chemical shifts for natural abun-
dance sites of N-αS and wt-αS at the protein/lipid
ratio used (1:250). Further, the INEPT polarization
Fig. 3. Dipolar-recoupled 13C NMR spectra of POPC/
EYSM/Chol raft-like membranes. (a) Site-resolved 2D
DROSSNMRspectraareshownforraft-likePOPC/EYSM/
Chol (1:1:1) lipid membranes at 48°C. Spectral planes
include unsaturated (115–135ppm), headgroup plus back-
bone(50–80ppm),andacylchain(0–40ppm)resonancesof
thephospholipidsandcholesterol.(b)Experimental 13C–1H
residualmagneticdipolarlineshapesandtheoreticalfitsare
showntogetherwith 13CNMRchemicalshiftprojectionsfor
raft-likemembranesandresonanceassignments(cf.Fig.2a).
provide a basis for interpreting the chemical shifts
andforassessingthemixingbehaviorofthelipidsin
the ternary system.40,41,50–55
In order to demonstrate the sensitivity of the 13C
NMR experiment to compositional phase behavior,
weshowinFig.4the13Cchemicalshiftspectrainthe
range15–45 ppmforthesesystems,corresponding
to the central bilayer region. Additional spectral
detailsforthemixedlipidsystemsarefoundinFigs.
S1–S3 of Supplementary Data, and tabulations are
given in Tables S1–S8. The binary EYSM/POPC Fig. 4. Isotropic 13C NMR chemical shift spectra
systemexhibitsaspectrumresemblingthesuperpo- demonstrate heterogeneity of raft-like membranes. 13C
sition of single-component spectra, consistent with INEPT-MAS NMR spectra are for single-component
the presence of demixed sphingolipid and glycer- POPC and EYSM, as well as mixtures of EYSM/Chol
olipid components.43 The binary POPC/Chol and (1:1), POPC/Chol (1:1), EYSM/POPC (1:1), and ternary
POPC/EYSM/Chol (1:1:1) mixtures obtained at 48°C.
EYSM/Chol spectra exhibit distinct polymethylene
regionsthatarisefromheterogeneousphospholipid– Note that the sum of binary spectra (1:1:1 linear
combination) reproduces the raft-like POPC/EYSM/Chol
phospholipid and phospholipid–cholesterol interac-
(1:1:1)ternaryspectrum.Thespectralagreementsupports
tionswithinthelosphingolipidandglycerolipidpools. the presence of cholesterol-enriched POPC and EYSM
Linearcombinationofthebinaryspectrareproduces domainsinthecompositionallyheterogeneousbilayer.
Annealingof Raft-Like Membranesvia α-Synuclein 2977
tentatively assigned to αS-bound and unbound
fractionsofthelipidpool.Anestimateofthelifetimes
for these two states is obtained from 1/Δδ = 26 ms,
whereΔδisthedifferenceinchemicalshiftofthetwo
resonance lines. The choline α carbon position is
proximal to the phosphate group and is the site of
phospholipaseDconversionofglycerophospholipids
tophosphatidicacidandcholine.Suchasite-specific
markeroftheinteractionofwt-αSwiththemembrane
maybeacorollaryofthephospholipaseDinhibitionby
αS shown previously in biochemical studies.59 How-
ever, this α-splitting does not occur with the N-αS
peptide, suggesting that interactions between the
protein and peptide with the membrane interfaceare
notequivalent,whichcanbeascribedtothereduced
binding partition coefficient of the small peptide
compared to the full-length protein.16 Differences in
the degree and lifetime of this specific interfacial
association can arise from contributions of the
unstructured C-terminus, additional lysine-enriched
repeatsofthewild-typeprotein,andthehydrophobic
sequence domain referred to as non-amyloid beta
component(residues61–95).Thelatterhasacritical
rolefornucleationoftheaggregationprocess,60which
has been shown in solid-state MAS NMR measure-
mentstoinvolvebindingtolipidmembranes.61
Fig. 5. SLF NMR reveals αS interactions with raft-like
lipid membranes. (a) 2D 13C chemical shift-dipolar Order parameters from solid-state
13C–1H NMR spectroscopy reveal membrane
coupling correlation spectra for raft-like POPC/EYSM/
Chol (1:1:1) membrane lipids containing wt-αS (protein/ perturbation by α-Synuclein
lipid molar ratio 1:250) at 48 °C. Spectral planes corre-
spond to unsaturated sites (115–135ppm), headgroup Fromthe2DDROSSspectra,weextractedslices
plus backbone (50–80 ppm), and saturated carbon seg- corresponding to RDC lineshapes of the chemically
ments of phospholipids and cholesterol (0–40ppm), and shifted resonance positions. The experimental and
exhibit pronounced differences compared to raft-like fitted RDC spectra in Fig. 6a and b are shown for
spectrainFig.3.(b)Isotropic 13Cchemicalshifts(below)
selected headgroup and acyl chain positions,
and13C–1Hdipolarlineshapes(above)extractedfrom2D
respectively. Note that the narrow RDC lineshapes
planes.ResonanceassignmentscorrespondtoFig.2a.
of the phosphocholine headgroup increase in
breadth in the presence of αS, while the acyl chain
RDCsdecrease.TheinterfacialRDClineshapesare
transferoftheDROSSpulsesequencewasfoundto well above the isotropic limit, and increases of the
be ineffective for the majority of cholesterol sites, RDCs indicate that the angular averaging of these
precluding meaningful analysis oftheseresonances. segmental positions is reduced, or that the average
Therefore,wefocussolelyonPOPCandEYSMinthis conformation is changed through interfacial associ-
system.Strikingdifferencesbetweenthe29–32 ppm ation. Another indication of this interaction is the
(CH ) region in the raft-like mixture alone (Fig. 3b) baseline oscillations of the headgroup RDC line-
2n
andinthepresenceofwt-αS(Fig.5b)areobserved.In shapes (Fig. 6a). Periodic artifacts arise from
this range, the chemical shift overlap is greatly truncationofthefreeinductiondecaypriortoFourier
reduced, giving a spectrum that resembles that transformationwhenlongspin–spinrelaxationtimes
recorded for the lipids in binary membranes (Fig. 4). of the segments are present. The long relaxation
Thesechemicalshiftchangesarenearlyidenticalfor times reflect greater isotropic motion of the head-
both the wt-αS and N-αS species (see Figs. S4 and groupsites.UponinteractionwithαS,thetruncation
S5, Tables S9–S12). Site-specific evidence for inter- oscillationsare diminished infrequency, suggesting
facial association is provided by a unique change at anincreasedlocal-fieldmagneticdipolarcontribution
the α position of the choline headgroup, common to to the transverse nuclear spin relaxation from
bothPOPCandEYSMphospholipids,inthepresence peptide and protein binding. The residual magnetic
ofthewt-αS(Fig.5b).Weobservetwopeaksat59.8 dipolar couplings of the raft-like membrane acyl
and59.5 ppm,suggestingtwomagneticallyorchem- chains without αS are characteristic of liquid-
ically distinct populations. The resonances may be ordered, cholesterol-rich membrane phases, as
2978 Annealingof Raft-Like Membranesvia α-Synuclein
and protein-induced changes to phospholipids that
may be probed in both the SLF and 2H NMR
experiments.28,62–64 In general, cholesterol order
parameters of ring carbons are also expected to
change as a function of membrane environment,
though in the DROSS experiment, we could not
accuratelymeasurethesevaluesduetoinefficiency
oftheINEPTpolarizationtransferatrigidcholesterol
sites. Focusing on the phospholipids, the absolute
13C NMR order parameter profiles of EYSM and
POPC show that in the presence of αS, the
interfacial |S | values increase while the acyl
CH
chain |S | values decrease. The observation of
CH
these changes substantiates that αS interacts with
thebiomembraneinterfaceandleadstodisordering
ofthehydrocarbonchains,oppositelytotheeffectof
cholesterol inthe raft-like system (see below).
To interpret the changes in |S | for the raft-like
CH
membranes,weobtainedtheabsoluteorderparam-
eter profiles for single-component and binary phos-
pholipid/phospholipid and phospholipid/Chol
membranes. The large differences of the POPC
and EYSM single-component order parameters are
due to the phase behavior and chemical properties
of the lipids (see Supplementary Data). Mixing of
POPCand EYSM causesan increase inthevalues
of|S |forPOPCandadecreasefor EYSM. Inthe
CH
single-component and binary POPC/EYSM disper-
sions, the S values indicate that the membrane
CH
lipidsareinthel phase.ForbinaryPOPC/Choland
d
Fig.6. Experimental 13C–1Hresidualdipolarcouplings EYSM/Chol membranes, cholesterol acts to con-
indicate wt-αS and N-αS interactions with raft-like mem- dense the lipids, yielding similar large order param-
brane phospholipids. (a) Experimental 13C–1H DROSS etersforbothPOPCandEYSMinl phases(Fig.7).
o
dipolarlineshapesandtheoretical fitsfor phosphocholine
The absolute order parameters for membrane lipids
α,β,andγheadgrouppositionsofPOPC(P)andEYSM(E)
in the raft-like ternary system are similarly large,
at 48°C inthepresenceof wt-αSand N-αS(protein/lipid
indicating cholesterol-enriched l lipid pools. Com-
molar ratio 1:250). The increase in RDCs for headgroup o
positionsisduetoαSinteraction.(b)RDCsofpalmitoyl(p), pared with these lipid membrane order parameter
oleoyl(o),fattyacyl(f),andsphingosine(s)chainsofPOPC profiles,thephospholipidchainsinraft-likemixtures
andEYSMinraft-likemembranesinthepresenceofwt-αS containingwt-αSandN-αSexhibitorderparameters
andN-αSat48 °C.Notethatfortheacylchains,thereisa trendingtowardslower|S |valuescharacteristicof
CH
decreaseinRDCsduetomembraneinteractionwithαS. the l phase. As noted above, this decrease is
d
opposite to the increase of phospholipid order
parameters caused by cholesterol. A possible
seen in corresponding solid-state 2H NMR studies cause of the change in order parameters is a
that afford higher resolution of individual segments disorderingoftheraft-likel membranehydrocarbon
o
withinthechainregion.30Incontrasttotheincrease environment, leading to an l -like phase, which is
d
of headgroup RDCs, for these chain positions, the also supported by the change in appearance of the
presenceofboththewt-αSproteinandN-αSpeptide chemicalshiftspectrumoftheternarylipidsystemin
leads to a reduction of the breadth of the RDC the presence of αS or N-αS. Thus, we propose that
linewidth, suggesting that disordering of the chains αS antagonizes the condensing and ordering effect
occurs. This hitherto unobserved change accompa- that cholesterol has on phospholipids through a
nyingthebindingofαSisaddressedindetailbelow. disorderingmechanismpresentedintheDiscussion.
To further characterize the structural perturbation The change of interfacial order parameters lends
at all resolved phospholipid positions and evaluate further insight into the disordering function of the
the local changes at the nonpolar hydrocarbon, protein. For the phosphocholine headgroup α, β, γ
backbone,andheadgroupregionscausedbyαS,we segments, the absolute order parameters increase
summarize in Fig. 7 the RDCs as order parameter in the presence of wt-αS and N-αS, indicating that
profiles of |S | versus carbon position. These both the N-terminal peptide and full-length protein
CH
profiles allow for site-specific evaluation of peptide interact with the membrane interface.16,58,65,66
Annealingof Raft-Like Membranesvia α-Synuclein 2979
Fig. 7. Segmental order profiles
characterize annealing of raft-like
phospholipidternarymixturebyαS.
Order parameters |S | (absolute
CH
magnitude)areplottedagainstcar-
bon position for (a) POPC and (b)
EYSM in (▼) ternary POPC/EYSM/
Chol(1:1:1),(◄)POPC/EYSM/Chol
(1:1:1) + N-αS, and (►) POPC/
EYSM/Chol + wt-αS membrane
mixtures at 48 °C (protein/lipid
molar ratio 1:250). Interfacial αS
associationwithlipidsitesproduces
large-scale structural changes
throughout the hydrophobic acyl
chainregion.Orderparametersare
compared to liquid-ordered (△)
phospholipid/Chol (1:1) mixtures.
The results support αS-induced
changes from liquid-ordered to liq-
uid-disorderedstates.
Moreover, the absolute order parameters for the characterized according to two structural quantities
glycerol sn-1, sn-2, and sn-3 positions (Fig. 7a), as derived from the segmental S order parameters
CH
wellastheresolvedsphingosineS4andS5segments obtained at uniquely resolved chemical shift sites
(Fig.7b),increaseinthe presenceofbothN-αSand of the phospholipid hydrocarbon chains using the
wt-αS.AsanestimateofthedepthofαSpenetration mean-torque model.31 The first quantity is the
into the membrane, the headgroup thicknesses for average cross-sectional area 〈A 〉 and the second
C
glycerolipids and sphingolipids are 9 Å and 7 Å, is the volumetric hydrocarbon thickness per phos-
respectively.Thesethicknessescanbeapproximate- pholipid chain D . For a given phospholipid type,
C
ly separated into phosphocholine (≈3–4 Å) and the value of 2D gives the overall thickness of the
C
backbone(≈7–9 Å)depths.Inthecaseofwt-αS,the membrane bilayer D , neglecting headgroup con-
B
change of order parameter is greatest at the zwitter- tributions D for the monolayer leaflets. In addition,
H
ionicphosphocholineheadgroup,suggestingthatthe the total interfacial cross-sectional area per phos-
proteinisapproximatelylocalizedtotheupperb3–4 Å pholipid may be estimated as 2〈A 〉 = 〈A〉, because
C
region of the bilayer. For N-αS, order parameters of order parameters of both chains at the membrane
the glycerol backbone and the segmental sites interface are treated equivalently in the NMR data
proximal to interfacial hydrogen bonding sites of reduction. Any perturbation affecting hydrogen
EYSM are affected more than the headgroup, bonding, electrostatics, or van der Waals interac-
indicating further penetration to b7–9 Å into the tions of the membrane lipids yields a change in
bilayer interface. Such changes at the l membrane S and will alter the structural quantities accord-
o CH
interfacemayinvolvedisruptionsofhydrogenbonding ingly. The 〈A 〉 and D values are presented in
C C
forEYSMandclosepackingoflipidsforbothPOPC Fig. 8a and b, respectively, for the αS-containing
and EYSM. These interactions contribute to the raft- raft-like mixtures, as well as the ternary raft-like
likeheterogeneityoftheternarymembrane,47–49and mixture at 48 °C (see Table S13). The 〈A 〉 and
C
their disruption can facilitate alteration of van der D results for binary l and l phases at 48 °C are
C o d
Waals hydrophobic contacts in the bilayer core also shown to assist in understanding the struc-
leadingtol states. tural perturbation of the raft-like membrane caused
d
by αS interaction.
The intrinsically disordered protein α-Synuclein By applying the mean-torque model,31 the cross-
remodels raft-like membranes containing sectionalareasdeterminedforeachphospholipidin
cholesterol theraft-likemembranemixturearefoundtobe〈A〉 =
57.6 Å2forPOPCand〈A〉 = 56.4 Å2forEYSM.The
Local molecular perturbations as discussed slightly smaller cross-sectional area for EYSM
above can also give rise to larger-scale changes reflects larger contributions to inter-lipid packing
in membrane structure. These changes may be duetohydrogenbondingandfavorablehydrophobic
2980 Annealingof Raft-Like Membranesvia α-Synuclein
backbone dimensions D , the mixed membrane
H
system has nonequivalent bilayer thickness contri-
butions of D ≈ 52.2 Å for POPC and D ≈ 45.8 Å
B B
forEYSM.Thisreflectsthelargerglycerolbackbone
thicknesscomparedwiththesphingosinebackbone,
aswellasthelonger18:1,cis-Δ9hydrocarbonchain
length of the oleoyl chain compared with the 16:1,
trans-Δ2 sphingosinechainofEYSM.Thesebilayer
thicknesses and cross-sectional areas point to an
equilibrium distribution of cholesterol-enriched, con-
densed complexes of lipids70 in liquid-ordered
phases,similartothebinaryphospholipid/cholester-
ol systems presented in Fig. 8, which possess
differentphospholipidhydrophobic thicknesses.
For raft-like membrane phospholipids in the pres-
enceofN-αS,thevalueofD forPOPCisreducedto
C
14.3 Å for the palmitoyl chain and 15.7 Å for the
monounsaturated oleoyl chain. Hydrocarbon thick-
nessesoftheEYSMfattyacylandsphingosinechains
are14.1 Åand13.8 Å,respectively.Thecorrespond-
ing bilayer thicknessestimates are found tobeD =
B
49.4 ÅforPOPCandD = 42.2 ÅforEYSMat48 °C.
B
Anoverallreductionofbilayerthicknessisaccompa-
nied by an increase in cross-sectional area per
phospholipid. We find that for POPC, the cross-
sectional area per phospholipid is 〈A〉 = 62.8 Å2 at
48 °C,whichissimilarto〈A〉 = 63.4 Å2inthecaseof
Fig. 8. Mean-torque model yields average cross-sec- EYSM. These relatively large values indicate that
tional areas and hydrocarbon thickness for POPC and peptide association with the membrane perturbs the
EYSM lipids showing αS-induced annealing of raft-like stabilizing interactions between lipids, giving rise to
membranes. (a) Average chain cross-sectional area 〈AC〉 thinnedbilayersanddisorderedlipids.
and(b)volumetrichydrocarbonthicknessDCforindicated Likewise, the wt-αS protein changes the mem-
mixtures at 48°C. Both D and 〈A 〉 are calculated for
C C brane thickness and cross-sectional area per phos-
individual acyl chains, that is, EYSM fatty acyl (N-
pholipidofPOPCandEYSM.At48 °C,thevaluesof
palmitoyl) and sphingosine chains, and POPC palmitoyl
D forthenonequivalentchainsofPOPCare14.3 Å
andoleoylchains.StructuralparametersforbinaryPOPC/ C
EYSM (1:1), POPC/Chol (1:1), and EYSM/Chol (1:1) (palmitoyl) and 15.7 Å (oleoyl), whereas values of
membranes support the proposed lo–ld structural change DCforEYSMare12.9 Åand12.5 Åforthefattyacyl
of ternary POPC/EYSM/Chol (1:1:1) membranes in the andsphingosinechains.ThebilayerthicknessD =
B
presence of wt-αS and N-αS. Thinning of D and an 49.4 ÅforPOPCisthesameasthatdeterminedfor
C
increase of 〈AC〉 in the αS-perturbed ternary system the N-αS system, while for EYSM, DB = 39.8 Å,
characterizethemembraneannealingprocess. indicating further perturbation. A cross-sectional
area for POPC of 〈A〉 = 62.8 Å2 is found, as in the
N-αS system, while the even more pronounced
matching between saturated chains. These cross- decreaseofEYSMbilayerthicknessisaccompanied
sectional areas per phospholipid compare closely by an increase of cross-sectional area to 〈A〉 =
with 2H NMR values determined for perdeuterated 69.4 Å2. These results point to an additional
sn-1 palmitoyl chains30 and also monolayer mea- interaction of the wt-αS protein with EYSM. This is
surementsobtained at30 mN/m surface tension for attributedtodifferencesinthebiophysicalproperties
thesameraftcomponents.67,68ForPOPCintheraft- of the glycerolipids and sphingolipids. One differ-
like system, D values of 15.6 Å and 17.1 Å are ence is the ability of wt-αS to undergo interfacial
C
found for the palmitoyl (16:0) and oleoyl (18:1, interactions at sphingosine backbone sites through
cis-Δ9) chains, respectively, at 48 °C (Fig. 8b). The electrostatics and hydrogen bonding. Such interac-
fatty acyl chain of EYSM (N-palmitoyl, 16:0, pre- tions are not available to POPC at the glycerol
dominant species ≈ 86%69) in the ternary mem- backbone. Nevertheless, the structural changes
branehasaD valueof15.9 Å.WecalculatedD for observed for both phospholipids show that signifi-
C C
the sphingosine chain assuming an effective 16:1, cantdisorderingofthebilayersoccurs.Largecross-
trans-Δ2 chain giving D = 15.4 Å at 48 °C in the sectional areas per phospholipid and small hydro-
C
ternary mixture. By considering symmetric bilayer carbon thicknesses are found that resemble the
leaflets, and including estimates of headgroup and binary POPC/EYSM membranes presented in
Annealingof Raft-Like Membranesvia α-Synuclein 2981
Fig. 8. The mechanism by which disruption of local Antagonism of α-Synuclein with cholesterol in
molecular sites gives rise to these structural raft-like membranes
rearrangements involves membrane annealing and
is discussed below. WeproposethatαScountersthecondensingeffect
of cholesterol within the raft-like membrane through
annealing of the lipid regions. This is a process
whereby a material is softened through external
Discussion
perturbation, leading to a lower energy state. Disrup-
tion of stabilizing lipid packing interactions promotes
Annealing of raft-based heterogeneity in lipid lateral lipid diffusion84 and disorders the membrane
membranes by α-Synuclein system. Such a rearrangement is suggested by the
increase in average cross-sectional area and reduc-
AmolecularunderstandingofParkinson'sdisease tionofhydrocarbonthicknessasthelipiddomainsare
requiresdelineationofαSinteractionswithbiomem- disrupted.Moreover,thechemicalshiftspectrareveal
branes implicated in the processes of neurodegen- a homogenization of hydrophobic environment of the
eration. The protein αS undergoes multiple binding membrane. We propose that the annealing likely
modes, principally initiated at the amphipathic N- involves αS–lipid interactions with the negatively
terminus,16,66withsmallunilamellarvesiclesthatare charged phosphodiester moiety of the zwitterionic
synaptic vesicle models,16,56,57,66,71–77 as well as phosphocholine headgroup and partially negative
largevesiclesthatmimictheplasmamembrane.73,78 hydrogen-bond acceptor sites of the sphingomyelin
In the physiological system, both of these neuronal backbone.Thesesitescanpromotetransientassoci-
membrane targets are highly heterogeneous,which ation of the protein and peptide with the membrane,
isattributedtodifferencesinmembranecomposition leadingtothesite-specific13CchemicalshiftandRDC
and phase.72,79–81 This heterogeneity is especially changesoftheheadgroupandbackbonesitesofthe
pronounced for raft-like mixtures where cholesterol phospholipids. Additional interactions with interfacial
interactsdifferentlywithPOPCandEYSM,48 result- hydrogenbondingcholesterolsitesmayplayarolein
ingincompositionallydistinctmicrodomains,albeitin the attraction of αS to the membrane interface, but
a similar ordered phase.43 Lateral compositional these effects are not directly observed in our experi-
heterogeneity gives rise to defects within the ments due to the inefficiency of the INEPT magneti-
membranethatfacilitateproteinbinding,asindicated zation transfer in the DROSS pulse sequence. We
by a membrane-initiated coil–helix transition of both have shown previously that amphipathic N-terminal
wt-αSandN-αS.16,18,19Thesedefectsexposehead- membrane binding initiates a protein conformational
group and backbone sites to solvent and protein.82 change.16OurNMRobservationsalsoshowthatwhile
The associated RDCs and S order parameter the N-αS peptide inserts to a greater extent into the
CH
values for headgroup and backbone sites for the membrane, wt-αS causes a more pronounced per-
POPC/EYSM/Chol (1:1:1) membrane mixture yield turbationoftheraft-likemembraneenvironment,due
strikingchangesthroughbindingofαStothebilayer. todifferencesinpartitioncoefficientofthetwospecies
The proposed restructuring of the raft-like mem- related to additional interaction sites on the wt-αS
branemixtureobservedusingsolid-state13CNMRis protein.Itispossiblethatdeepinsertionofpeptidesor
shown schematically in Fig. 1. Here, EYSM and proteins within the hydrocarbon bilayer may cause
POPC exist in condensed ordered regions in increases of segmental order and lead to erroneous
association with cholesterol. In the raft-like mem- structural conclusions regarding bilayer integrity.
branemixture,interfacialinteractionsbetweenlipids However, in our measurements, we resolve an
and cholesterol prevent lateral diffusion and self- increase of headgroup and backbone order param-
mixing of lipids,83 leading to a compositionally eters while hydrocarbon chain order reduces upon
heterogeneous lateral distribution in so-called do- peptide and protein interaction. Therefore, further
mains.Nanometer-scaledemixingoflocallyenriched structural analysis using these hydrocarbon chain
POPC/Chol and EYSM/Chol ordered complexes, order parameters is warranted. The mean-torque
which is unresolved in fluorescence microscopy of results provide estimates of the bilayer structural
thisraft-likesystem,43isindicatedbydifferentvalues dimensions, thereby identifying a striking shift of
of the hydrocarbon thickness and average cross- coexistingl phaseregionstoamorel -likephasein
o d
sectionalareaofthephospholipids. Thisseparation the raft-like membrane. Such effects are not
isattributedtolocaldifferencesininterfacialelectro- unexpected, since as an amphipathic protein16,85
statics,hydrogenbonding,anddifferencesinhydro- αS shares this feature in common with various
phobic volume and length between the membrane antimicrobialpeptides.62Thesepeptidesalsoperturb
components, for example, hydrocarbon mismatch. the membrane interface and induce changes in the
Interfacial defects between these complexes likely hydrophobic membrane center.86–89 In general, the
involvemixedPOPC/EYSM/Cholregionsasindicat- disruption of stabilizing interfacial interactions and
edinFig.1. compositional homogenization enables membrane
2982 Annealingof Raft-Like Membranesvia α-Synuclein
thinning,withaconcomitantincreaseofphospholipid drophobic acyl chain region. These physical changes
cross-sectionalareathatcanmodulatespontaneous maymodulatefusion,eitherdirectlythroughchangesin
membrane curvature.90–93 While these remodeling the membrane strain and lipid distribution91,97 or
propertiesofthemembranearenotspecifictoαS,itis indirectly by altering lateral lipid mobility that in turn
likelythat inthe context of synaptic membranes and influences membrane protein localization and organi-
neurotransmission, such interactions may play a zation. In this context, the inherent flexibility of αS
significantrole. interaction with biomembranes can be altered by
The site-specific 13C NMR results show that protein aggregation into toxic oligomeric species,
annealing in raft-like membranes by αS involves eventuallyleadingtoimpairmentofneuronalsignaling.
interfacial lipid interaction and removes so-called The relation of protein plasticity and biomembrane
defects. This is consistent with the hypothesis that remodeling is an important aspect that can aid in
αS eliminates interfacial fusion sites associated with understanding neurological dysfunction implicated in
compositional heterogeneity, thus reducing the prob- the etiology of Parkinson's disease. How such
abilityoffusion events.Thisfunction issuggestedby membrane-dependent mechanisms are related to the
ourinvitroresultsandisdemonstratedinrecentinvivo aggregation propensity of αS and neurodegeneration,
studies, where the inhibition of raft-like mitochondrial andwhethersimilarmechanismsareoperativeinother
membrane fusion has been shown in cultured cells neurodegenerative disorders such as Alzheimer's
andCaenorhabditiselegans.7 Additionalbiochemical disease, are important topics for future research.
evidencesuggestingthatαSplaysaroleinexocytotic
membrane fusion comes from the observation of
Materials and Methods
specific association of the C-terminus of αS with a
criticalsubunitoftheSNAREfusioncomplex(synap-
tobrevin),inconjunctionwithαSN-terminalmembrane Samplepreparation
interaction.94 The protein–lipid interaction assists in
SNARE complex assembly,94 thereby facilitating POPC and EYSM [predominant species N-(palmitoyl)-
subunit tethering to lipid vesicle membranes. The sphing-4-enine-1-phosphocholine] were from Avanti Polar
process is highly dependent on the presence of Lipids Inc. (Alabaster, AL). Cholesterol was procured from
arachidonic acid,95 a polyunsaturated fatty acid Sigma-Aldrich(St.Louis,MO).Monomericwt-αSwasagift
from Drs. Frits Kamp and Christian Haass, University of
precursor to many intracellular and extracellular
Münich, Germany, and the N-αS peptide (1–25) was from
signaling molecules. It is interesting to note that the
Primm Biotech, Inc. (Cambridge, MA). Multilamellar lipid
plasma-membrane-associated SNARE fusion com-
vesicledispersionswerepreparedfromlyophilizedpowder
plexrequiresraft-likemembranesenrichedincholes- hydrated with 2H O at pH≈7 (Cambridge Isotopes, Cam-
terolandsphingomyelin.13,15Localizationandfunction bridge,MA).Addit2ionalbufferingwasnotused,thusavoiding
oftheproteincomplexaredeterminedbythebalance salt-screeningeffectsontheαS–lipidinteractions.Peptides
ofliquid-disorderedandliquid-orderedphases96thatin andproteinswereco-addedatalow1:250molarratioofN-
lightofourresultscanbemodulatedbyαSbinding. terminalequivalentsofαStototallipid,soastolimitprotein
aggregation and study changes of the lipid membranes
Conformational plasticity of α-Synuclein in causedbytheproteininitsmonomericstate.Wehaveshown
previously16thatbindingoftheproteinandoftheN-terminal
neural function and dysfunction
peptidetosmallunilamellarvesiclesissaturated(99%and
97%, respectively) at a total molar ratio of 167 lipids per
Importantly, the conformational plasticity of αS is
protein (peptide). A direct determination of thermodynamic
likely to be critical to its function in membrane lipid bindingconstantsusingmultilamellarsystemsandthe1:250
fusion and neurodegeneration. Fusion events are protein/lipidratiodescribedinthisarticlewasnotfeasible,but
highly dynamic, whereby the protein is required to is likely on the order of that observed previously. The
respond rapidly and reversibly to changes in multilamellar vesicle dispersion was then subjected to 3–5
membrane phase,19 shape,78 and electrostatic freeze–thaw–mixing cycles. Lipid samples were tested for
environment.16 Such interactions have been identi- ester hydrolysis before and after the experiments by thin-
layer chromatography with CHCl /MeOH/H O (65:30:5),
fied as contributing to the biasing of conformational 3 2
ensembles of αS. Our results reveal membrane followedbycharringwith40%H2SO4inEtOH.
perturbations caused by αS that further emphasize
Solid-state NMRspectroscopy
the importance of raft-like, compositionally hetero-
geneous membranes as an important target for this
intrinsically disordered protein.11 Not only are the Solid-state MAS NMR experiments were conducted
using a narrow bore 11.7-T AVANCE-I spectrometer
structural states of the protein perturbed in this
system (Bruker BioSpin Corporation, Billerica, MA). The
interaction, but the properties of the membrane are
SLF experiment DROSS22 was implemented with the
alsochanged.Wefindthatsuchaninteractionoccurs
Bruker Topspin software platform. A triple-channel MAS
with raft-like membrane mixtures through specific NMRprobe(DSI-733;DotyScientificInc.,Columbia,SC)
associationwithinterfaciallipidsites,whichpropagates was used for all experiments. Samples were loaded in
large-scale structural deformation throughout the hy- 40-μLsealingcellsandplacedin4-mmthin-wallzirconium