Fibrillogenic and Non-fibrillogenic Ensembles of SDS ... - CCMB

doi:10.1016/j.jmb.2006.09.085 J. Mol. Biol. (2006) 364, 1061–1072Fibrillogenic and Non-fibrillogenic Ensembles ofSDS-bound Human α-SynucleinMd. Faiz Ahmad, Tangirala Ramakrishna, Bakthisaran Raman⁎ andCh. Mohan Rao⁎Centre for Cellular andMolecular Biology, Hyderabad500 007, India*Corresponding authorsFibril formation of α-synuclein is associated with several neurodegenerativediseases, including Parkinson's disease in humans. The anionic detergentsodium dodecyl sulfate (SDS) can accelerate the fibril formation in vitro.However, the molecular basis of this acceleration is not clear. Our studyshows that native α-synuclein exhibits relatively less fibril growth despiteproviding fibril seeds for nucleation. The presence of SDS promotes theseeded fibril growth in a concentration-dependent manner, with an optimalconcentration of 0.5–0.75 mM. We used isothermal calorimetry, hydrophobicdye binding and circular dichroism spectroscopy to characterize theprotein–detergent interactions as a function of the concentration of SDS.Interaction of SDS with α-synuclein when studied by isothermal titrationcalorimetry and hydrophobic dye-binding reveals a similar characteristicoptimal behavior between 0.5 mM and 0.75 mM SDS. The study shows twotypes of ensembles of α-synuclein and SDS: the fibrillogenic ensemblesformed with optimal concentration of SDS around 0.5–0.75 mM arecharacterized by enhanced accessible hydrophobic surfaces and extendedto partially helical conformation, while the less or non-fibrillogenicensembles formed above 2 mM SDS are characterized by less accessiblehydrophobic surfaces and maximal helical content. Little or no fibrillogenicityof the ensembles observed above 2 mM SDS could be partly because ofthe observed intrinsic instability of the fibrils under the condition.© 2006 Elsevier Ltd. All rights reserved.Keywords: α-synuclein; SDS; Parkinson's disease; amyloid; isothermaltitration calorimetryIntroductionAggregation of α-synuclein into filaments orfibrils is involved in some neurodegenerativediseases such as Parkinson's disease, multiplesystem atrophy and other synucleinopathies,including dementia with Lewy bodies. 1–5 Humanα-synuclein is a 140 amino acid residue protein withnatively unfolded conformation, 6,7 abundantlyexpressed in the brain, where it is concentrated inpresynaptic nerve terminals. 8–10 The mechanism offibril formation of α-synuclein and the factorsAbbreviations used: SDS, sodium dodecyl sulfate; bis-ANS, 1,1′-bis (4-anilino) naphthalene-5,5′-disulfonic acid;ThT, thioflavin T; ITC, isothermal calorimetry.E-mail addresses of the corresponding authors:raman@ccmb.res.in; mohan@ccmb.res.incontributing towards its fibril formation have beena subject of intense research over the years.Recombinant human α-synuclein has been shownto form fibrils in vitro under physiologically relevantpH conditions, but the reaction requires a high concentrationof protein, vigorous shaking or agitationand takes a long time, often days to weeks. 11,12However, fibril formation can be accelerated byacidic pH, 11 point mutation, 13–15 truncation, 16 andby additives such as glycosaminoglycans, 17 polyamines,18 and anionic detergents and fatty acids. 19–22α-Synuclein has been shown to be present insynaptic vesicles, 23 and to interact with lipidmembranes. 24–28 A recent study shows that α-synuclein has a role to play in synaptic function. 29Several groups have investigated its interaction withlipid membranes 24–28 and detergent micelles 19,30–32to understand the role of such interactions in thefibril formation of the protein. However, the role oflipid membranes in the fibril formation remains0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.

1062 SDS-induced Fibril Growth of a-Synucleincomplex and debatable. Two schools of thoughtsexist: one contends that membrane favors aggregationand fibril formation, 20,33,34 while the othercontends that membrane disaggregates and inhibitsfibril formation. 28,35,36 Zhu and Fink have reportedthat the α-helical conformation induced by theanionic lipid vesicles is not fibrillogenic. 28 Fromthe forgoing, it is clear that the role of membranes ormembrane mimetic conditions in α-synuclein fibrilformation is not yet clearly understood. Unequivocalunderstanding of the role of membraneenvironment in the fibril formation of α-synucleinis important in understanding synucleinopathies.The anionic detergent, sodium dodecyl sulfate(SDS) has been used in some studies to understandthe role of a membrane-mimetic environment in thestructure and fibrillogenic properties of α-synuclein.19,30,31 The presence of SDS has been shown toaccelerate fibril formation. 19,37 Various structuralstudies have used very high concentrations of SDS(at which it forms micelles) to determine the SDSinducedfolded conformation of α-synuclein. Thefibrillogenicity of such a folded conformation is notclear. We set out to systematically compare theseeded fibril growth of α-synuclein as well as SDS–αsynucleininteractions as a function of the concentrationof SDS. In contrast to the rapid fibril growthobserved earlier with other fibrillogenic polypeptidesor proteins such as amyloid β peptides 38 or β2-microglobulin 39 upon seeding, our present studyshows that α-synuclein, though natively unfolded orintrinsically unstructured, 6,7 exhibits subtle differencesin the seeded fibril growth. Our isothermaltitration calorimetry (ITC), circular dichroism (CD)spectroscopy and 1,1′-bis (4-anilino) naphthalene-5,5′-disulfonic acid (bis-ANS) binding studies alongwith studies on the seeded fibril growth show thepresence of two types of ensembles of SDS–αsynuclein.These two ensembles show interestingdifferences, including fibrillogenicity.ResultsDual behavior of SDS in the fibril growth ofα-synucleinWhen native α-synuclein in buffer alone wasincubated (with no stirring or agitation) with fibrilseeds, no increase in thioflavin T (ThT) fluorescencewas observed (Figure 1(a)). Prolonged incubation(34 h) did not yield a significant increase in ThTfluorescence (data not shown). Wood et al. havereported that α-synuclein fibrillogenesis is nucleation-dependent.40 Their results show that the fibrillationoccurs only after prolonged incubation (a fewdays) even in the presence of seeds. In comparison,other nucleation-dependent, fibril-forming systems,such as amyloid β peptides 38 or β 2 -microglobulinunder acidic conditions, 39 exhibit rapid fibril growth(in minutes) once seeded. This implies that α-synuclein exhibits subtle differences in its nucleationFigure 1. SDS-perturbed state, but not native humanα-synuclein, is compatible with fibril growth. (a) SDSinducedfibril growth of α-synuclein monitored by ThTfluorescence. α-Synuclein (1 mg/ml) in 20 mM Hepesbuffer (pH 7.0) was incubated at 37 °C with 0.1 mg/ml offibril seed in the absence (open circles) and in the presence(filled circles) of 0.15 mM SDS and the sample of α-synuclein (1 mg/ml) in the presence of 0.15 mM SDSincubated without fibril seeds (open triangles). The insetshows transmission electron microscopic image of fibrilsof α-synuclein formed in the presence of both the fibrilseed and SDS. (b) Association-induced β-sheet structureupon fibril growth of α-synuclein studied by far-UV CDspectroscopy. Far-UV CD spectra of the samples of α-synuclein (1 mg/ml) in the presence of 0.15 mM SDSincubated at 37 °C for 4 h in the absence (curve 1) and inthe presence of 0.1 mg/ml fibril of seed (curve 2). The far-UV CD spectrum (curve 3) of the sample of α-synucleinincubated with only fibril seed (without SDS) is shown.[θ] MRM is the mean residue mass ellipticity.dependency; nucleation alone is not sufficient for therapid formation of α-synuclein fibrils.Anionic amphiphilic molecules such as SDS andfatty acids are known to induce fibril formation of α-synuclein. 19–22 We have studied the seeded fibrilgrowth of α-synuclein in the presence of 0.15 mMSDS and found that ThT fluorescence intensityincreased as a function of time and was saturatedby 4 h (Figure 1(a)), indicating that fibril growthoccurs rapidly. Electron microscopic examination ofthe sample showed fibrillar morphology (inset inFigure 1(a)). Incubation of α-synuclein in thepresence of SDS without the seed did not result inincrease in ThT fluorescence over the observedperiod of time (Figure 1(a)).

SDS-induced Fibril Growth of a-Synuclein1063Ordered aggregation of proteins to amyloid fibrilsis known to be associated with cross β-sheetstructure. 41 We have investigated the generation ofβ-sheet structure upon fibril growth of α-synucleinby far-UV CD spectroscopy. Consistent with theresults of ThT-binding, the association-inducedβ-sheet generation is observed only with samplesof α-synuclein incubated with both SDS and thefibril seed (Figure 1(b)). The small extent of β-sheetstructure indicated by the spectrum of the sample ofnative α-synuclein incubated with seed was contributedby the seed and was not due to the fibrilgrowth, as judged by the far-UV CD spectrum ofseed alone (spectrum not shown).Figure 2 shows the extent of fibril growth of theprotein as a function of the concentration of SDS asmeasured by ThT fluorescence. The extent of fibrilgrowth increased dramatically and attained amaximum at a concentration of 0.5 mM SDS; itdecreased progressively with further increase in theconcentration of SDS. Fibril growth did not occur atconcentrations of SDS above 2 mM. One possibilityfor this biphasic nature of the profile is that theprotein to detergent ratio with some stoichiometry isimportant in the fibril formation of α-synuclein.Even when two different concentrations of α-synuclein(1 mg/ml or 0.22 mg/ml) were used, theoptimal concentration of SDS for fibril growthremained around 0.5 mM (see Figure 2 (a) and(b)). However, the width of the profile is relativelysharper at the lower concentration of proteincompared to that at the higher concentration.Thus, all our results show that SDS exhibits dualbehavior in the fibril formation of α-synuclein. Inorder to understand the dual behavior of SDSinducedfibrillogenicity, we have further studied theprotein–SDS interactions by ITC, hydrophobic dye,bis-ANS binding, time-resolved fluorescence spectroscopyand CD.Interaction of SDS with α-synuclein studied byisothermal calorimetryFigure 3(a) illustrates an ITC experiment wherewe have injected 10 μl of 7 mM SDS repeatedly intothe reaction cell containing 15 μM α-synuclein.Relatively low and constant exothermic heat flowwith the enthalpy change, comparable to theenthalpy change of dilution of the detergent(blank), was observed in the initial few additionsof the injectant (up to a certain concentration ofSDS). Further increase in the concentration of SDSled to a large exothermic enthalpy change, reachinga maximum at 0.5 mM SDS, which decreased uponfurther increase in the concentration of SDS. Thisinteresting ITC profile of SDS–synuclein interactionmight be described in two parts: the first part mightbe largely driven by entropy, with little or no changein enthalpy, thus showing no observable heattransfer in the first few injections; after saturatingthis interaction, further injections might lead to asecond part of the interaction that is driven byenthalpy, as seen by significant exothermic heattransfer. If such a description was true, altering theconcentration of synuclein should shift the profilesignificantly. Figure 3(b) shows the ITC profile withonly 5 μM α-synuclein. Despite such a large decreasein the concentration of α-synuclein, the ITC profiledid not show a shift to lower concentrations of SDS(maximum is ∼0.65 mM SDS). Moreover, when weincreased the concentration of α-synuclein to 70 μM,the ITC profile still exhibited a peak around 0.5 mMSDS. The peak, however, was broad, taperinggradually beyond 2 mM SDS (data not shown).Thus, the ITC profile cannot be used to derivestoichiometric thermodynamic parameters. However,the study shows a characteristic optimumaround 0.5 mM SDS (which parallels the fibrillogenicity),suggesting formation of some non-stoichiometricensembles of the protein with thedetergent.Fibrillogenic ensembles of α-synuclein with SDSexhibit enhanced hydrophobic surfacesFigure 2. SDS-induced fibril growth of human α-synuclein as a function of SDS concentration monitored byThT fluorescence. α-Synuclein at (a) 1 mg/ml or (b)0.22 mg/ml in Hepes–NaOH buffer (pH 7.0), wasincubated with 10% fibril seed at 37 °C for 4 h at variousconcentrations of SDS.Accessible hydrophobic surfaces on proteins canbe studied using the fluorescent hydrophobic probebis-ANS. 42 The probe is non-fluorescent in a polarenvironment. Upon binding to hydrophobic surfaces,its fluorescence increases accompanied by ablue shift in its emission maximum. 42 bis-ANS bindsto native α-synuclein and exhibits enhanced fluorescence(Figure 4(a)). The fluorescence intensity

1064 SDS-induced Fibril Growth of a-SynucleinFigure 3. Isothermal titration calorimetry showing a cooperative exothermic reaction between human α-synucleinand SDS at 30 °C. (a) Titration of 15 μM (0.22 mg/ml) α-synuclein with 7 mM monomeric SDS. (b) Titration of 5 μM(0.072 mg/ml) α-synuclein with 7 mM monomeric SDS. Titrations were performed by adding aliquots of 10 μl each time.Both the protein and the detergent were buffered at pH 7.0 with 20 mM Hepes–NaOH. The thick line in the dQ/dt (rate ofenthalpy change) window and filled circles in Q (enthalpy change) window represent the observed heat flow upontitration of the protein with the detergent. Thin line in the dQ/dt window and open circle in the Q window represent theblank titration (the heat flow of the dilution of the detergent to buffer alone).increases further upon addition of 0.25 mM, 0.5 mMand 1.0 mM SDS, and then decreases at 2 mM SDS.bis-ANS in 10 mM SDS without the protein shows anegligibly low level of fluorescence. Figure 4(b)further explores this biphasic behavior; the plot offluorescence intensity versus the concentration ofSDS peaks at 0.75 mM SDS and decreases graduallywith increasing concentration of SDS. This resultshows an increase in the accessible hydrophobicsurfaces of the protein or the protein–detergentensembles up to 0.75 mM SDS. The emissionmaximum red shifts slightly below 2 mM SDS andin a more pronounced manner above 2 mM SDS(Figure 4(b) inset). The minor red shift (below 2 mMSDS), despite an increase in the fluorescenceintensity, indicates that the intrinsic hydrophobicityof at least some sites decreases or some relativelymoderate hydrophobic sites become accessible. Thedecrease in the fluorescence intensity accompanyingthe pronounced red shift above 2 mM SDS reflects adecrease in the accessible hydrophobic surfaces.Similar results were obtained with 0.22 mg/ml of α-synuclein (Figure 4(c)). Thus, our results demonstratesubstantial rearrangement of hydrophobicsurfaces of the protein–detergent ensembles, whichmay be important for fibrillogenicity.The fluorescence life-time of the polarity-sensitivefluorophore ANS has been used to study conformationalchanges and partially unfolded states ofproteins; 43,44 it exhibits longer lifetimes upon bindingto protein hydrophobic surfaces. We have usedtime-resolved fluorescence spectroscopy to study thebis-ANS binding to α-synuclein. The fluorescencedecay profile of the sample of bis-ANS bound tonative α-synuclein (Figure 5) can be fit with a threeexponentialdecay model, with lifetimes τ1, τ2 and τ3of around 0.15 ns, 2.6 ns and 7.6 ns, respectively. Thefast decay component (τ1, 0.15 ns) could representunbound bis-ANS, while τ2 and τ3 represent thepopulations of bis-ANS bound to at least two types ofhydrophobic surfaces of α-synuclein.The fluorescence decay profiles of bis-ANS in thesample of α-synuclein with increasing concentrationsof SDS could also be fit with a threeexponentialdecay model. The fast component, τ1,remains unchanged. Remarkable changes could beobserved in the lifetimes of τ2 and τ3, as well as intheir relative amplitudes as a function of theconcentration of SDS. τ2 increases from 2.6 ns inthe absence of SDS to almost 3.5 ns in the presence of0.5 mM SDS (Figure 6(a)). With further increase inthe concentration of SDS, τ2 decreases progressively.On the other hand, τ3 drops sharply from7.6 ns in the absence of SDS to

SDS-induced Fibril Growth of a-Synuclein1065predominantly binding to and reporting the hydrophobicsurface of the protein component. (1) Thefluorescence intensity of bis-ANS is not enhancedsignificantly by SDS alone. (2) The emission maximum,even in the presence of 20 mM SDS (in micelleform), was found to be 510 nm, which is significantlydifferent compared to the probe bound to α-synuclein alone (486 nm) or to the protein–detergentcomplex (486–496 nm, depending on the concentrationof SDS as shown in the inset in Figure 4(b)). Evenin the presence of 20 mM SDS, the fluorescenceintensity of bis-ANS was found to be less than sevenarbitrary units compared to a high value rangingfrom 20–75 arbitrary units observed in the presenceof α-synuclein alone or both the protein and SDS(Figure 4(b)). (3) The decay profile of the probe in thepresence of the SDS micelle could be fit with a twoexponentialdecay model, with the population havingan average lifetime of 1.97 (with standard error(S.E.) of 0.005, n=4) contributing 90% (S.E.=1.07) ofthe fluorescence intensity representing the micelleboundbis-ANS molecules. The population having alifetime of 0.14 ns (S.E.=0.04) contributing 9.4%(S.E.=1.07) fluorescence intensity may represent theFigure 4. Binding of the hydrophobic fluorescentprobe, bis-ANS to the ensembles of α-synuclein andSDS. (a) Fluorescence spectra of 5 μM bis-ANS bound to1 mg/ml of α-synuclein in the absence and in the presenceof SDS (concentrations as indicated). The fluorescencespectrum of bis-ANS in the presence of 10 mM SDS aloneis also shown (curve a) (b) Variation of fluorescenceintensity at the emission maximum of bis-ANS when1 mg/ml of α-synuclein was titrated with increasingconcentrations of SDS. The inset shows the change in thewavelength of emission maximum as a function of theconcentration of SDS. (c) Variation of fluorescenceintensity at the emission maximum of bis-ANS when0.22 mg/ml of α-synuclein was titrated with increasingconcentrations of SDS. The samples were excited at390 nm with excitation and emission band passes set at5 nm; the buffer was 20 mM Hepes–NaOH (pH 7.0).Thus, these results show a significant reorganizationof hydrophobic sites, which may represent eitherchange in the conformation and polarity of theexisting sites or generation of new surfaces upon α-synuclein–detergent interactions.It is possible that bis-ANS binds to the protein or tothe detergent component of the ensembles. Thefollowing observations suggest that bis-ANS isFigure 5. Time-resolved fluorescence measurement ofbis-ANS bound to α-synuclein. (a) Fluorescence decay ofbis-ANS bound to α-synuclein in 20 mM Hepes–NaOHbuffer (pH 7.0), could be fit with a three-exponential decaymodel. The goodness of the fit was assessed by the χ 2 test,which gave a value close to 1, and the weighted residualsare shown in the lower panels of the Figure. Arepresentative decay profile out of four experimentaldata is shown in each case. The insets show mean lifetimes(τ) in nanoseconds and pre-exponential amplitude (α) inpercentage with the standard errors indicated within theparentheses. bis-ANS was excited at 337 nm using apicosecond LED light source.

1066 SDS-induced Fibril Growth of a-SynucleinSDS-induced secondary structural generationand fibrillogenicity of α-synucleinFigure 7 shows the change in the secondarystructure of α-synuclein from an unstructuredconformation to an α-helical conformation uponinteracting with SDS. The negative ellipticity at222 nm, and hence the helical content, increasesprogressively as the concentration of SDS increasesto 2 mM, and becomes constant with furtherincrease in the concentration of SDS. The SDSinducedfolding of α-synuclein to an α-helicalstructure is known. 30,45,46 However, the fibrillogenicnature of such an SDS-induced folded structure wasnot investigated. We have compared the extent ofSDS-induced folding with SDS-induced exposure ofhydrophobic surfaces and fibril growth. Maximallyα-helically folded α-synuclein in the protein–detergentensembles above 2 mM SDS, as judged byellipticity at 222 nm (Figure 7), exhibit relatively lessexposed hydrophobic surfaces (Figure 4) and do notexhibit fibrillogenicity (Figure 2). However, partiallyfolded species of α-synuclein in protein–detergentensembles below 2 mM SDS exhibit highly exposedhydrophobic surfaces and are fibrillogenic.Dual behavior of SDS in fibril formation and fibrildepolymerization of α-synucleinThe reason for poor fibril growth of α-synucleinabove 2 mM SDS could either be the conformationalconstraint in the protein–detergent ensembles, suchthat it is not competent for fibril growth, or therelative fibril instability under such conditions, orboth. We have measured the fibrils using ThTbindingafter incubating fibrils of α-synuclein(0.2 mg/ml with respect to α-synuclein concentration)at different concentrations of SDS for 1 h at37 °C (Figure 8(a)). Diluting the SDS (to approxi-Figure 6. Variation of fluorescence lifetimes of bis-ANS bound to α-synuclein as a function of the concentrationof SDS. Dotted vertical lines approximately divide thepopulations of fibrillogenic and less or non-fibrillogenicensembles of α-synuclein and SDS. Traces 1 and 2 in therelative amplitude panel correspond to the lifetimes τ2and τ3, respectively.unbound probe. Thus, fluorescence lifetimes of bis-ANS bound to α-synuclein and the SDS micelle aresignificantly different. Since we could not observe afluorescence lifetime component corresponding tothe lifetime of the probe in the micellar environment(around 1.9 ns) at any concentration of SDS in thepresence of protein (Figure 6), the changes in thefluorescence lifetimes of bis-ANS are likely to reportconformational changes associated with hydrophobicsurface alteration in the protein component of theprotein–detergent ensembles.Figure 7. SDS-induced folding of α-synuclein monitoredby far-UV CD spectroscopy. Variation of the meanresidue mass ellipticity ([θ] MRM ) at 222 nm of α-synuclein(1 mg/ml) as a function of the concentration of SDS. Inset:far-UV CD spectra of α-synuclein in 20 mM Hepes–NaOHbuffer (pH 7.0) in the absence (curve 1) and in the presenceof 0.15 mM SDS (curve 2), 0.5 mM SDS (curve 3), 1 mMSDS (curve 4) and 2 mM SDS (curve 5).

SDS-induced Fibril Growth of a-Synuclein1067from β2-microglobulin (referred to as K3) underacidic conditions. 47 We have tested if amorphousaggregation occurs with α-synuclein under ourexperimental conditions. Light-scattering of α-synucleindid not change significantly with increasingconcentration of SDS (Figure 9(a)). Since the multimericassembly of αB-crystallin (∼550 kDa) isknown to be sensitive to SDS, 48 we have monitoredthe SDS-induced dissociation of the protein forcomparison (Figure 9(a)). The scattering value ofα-synuclein in the absence or in the presence of SDSis far lower than that of αB-crystallin, indicating theabsence of even soluble large oligomers of α-synuclein; if present, their population is not highenough to be detected. Under similar conditions, theDTT-induced aggregation of insulin gives a verylarge scatter value (approximately 250 arbitraryunits, data not shown). All these results show thatSDS does not induce amorphous aggregation of α-synuclein significantly.Figure 8. SDS concentration-dependent fibril dissociationof α-synuclein. (a) Fibrils of α-synuclein present afterincubation of 0.2 mg/ml of fibrils in 20 mM Hepes–NaOHbuffer (pH 7.0) at 37 °C for 1 h as a function of theconcentration of SDS determined by ThT binding. Theinset shows the time-dependent dissociation of the fibrilswhen incubated in the presence of 1 mM (open circles),2 mM SDS (filled circles) and 5 mM SDS (filled triangles).(b) β-Sheet to α-helical conversion of the fibrils of α-synuclein upon its dissociation in the presence of highconcentrations of SDS. Far-UV CD spectra of the fibrils ofα-synuclein incubated at 37 °C in the presence of 0.5 mMSDS (curve 1) and in the presence of 4 mM SDS at 30 min(curve 2) and at 60 min (curve 3) of incubation.mately 15 μM) due to the dilution of the fibrils stockresults in significant amount of dissociation and lossof the fibrils. The fibrils are significantly stable up toa concentration of SDS of 1 mM. However, increasingthe concentration of SDS further leads to drasticloss of fibrils due to dissociation. The dissociation isassociated with conversion of β-sheet structure to α-helical structure, as revealed by far-UV CD spectroscopy(Figure 8(b)). This result shows that fibrilinstability is one of the factors contributing to theobserved lack of fibril growth above 2 mM SDS.Treatment with SDS does not lead to amorphousaggregation of α-synucleinA recent study by Yamaguchi et al. has shown thatamorphous aggregation competes with SDSinducedfibril formation of a small peptide derivedFigure 9. Measurements of light-scattering and criticalmicellar concentration. (a) Light-scattering (at 465 nm) ofsamples of 0.3 mg/ml of α-synuclein (filled circles) and0.3 mg/ml αB-crystallin (filled triangles) upon treatingwith increasing concentrations of SDS; the buffer was20 mM Hepes–NaOH (pH 7.0). The light scattering of thebuffer alone with increasing concentrations of SDS (opencircles) is also shown. (b) The critical micellar concentrationof SDS in 20 mM Hepes–NaOH buffer (pH 7.0)measured using 10 μM diphenylhexatriene as the probe.The fluorescence intensity of the probe at 430 nm withincreasing concentrations of SDS in the absence (opencircles) and in the presence (filled circles) of 0.3 mg/ml ofα-synuclein is shown. The probe was excited at 358 nm.The broken line indicates a slope change in the profile offluorescence intensity versus concentration of SDS correspondingto the α-synuclein sample.

1068 SDS-induced Fibril Growth of a-SynucleinCritical micellar concentration of SDS in theabsence and in the presence of α-synucleinAn earlier study by Yamamoto et al. showedthatβ2-microglobulin exhibits optimal concentrations ofSDS around 0.5 mM in its SDS-induced fibrilformation and proposed that fibril formation occursnear the critical micellar concentration (cmc) ofthedetergent in the presence of the protein. 49 Wemeasured the cmc of SDS in our buffer system usingdiphenylhexatriene as a probe. The fluorescence of theprobe is low below the cmc and increases dramaticallyabove the cmc. 50 As this probe is uncharged, thismethod avoids interference from detergent charge indetermining cmc. 50 Figure 9(b) shows that the cmc ofSDS is around 7.5 mM in the buffer. In the presence ofα-synuclein, the fluorescence intensity increases as afunction of the concentration of SDS without a distinctinflection point, in contrast to that observed in theabsence of the protein. However, there is a slopechange around 0.5 mM SDS. Considering the increasein the fluorescence intensity even before 0.5 mM SDS,it is not clear whether this point represents the cmc.However, it is evident that the presence of the proteinalters the property of the detergent. Thus, our studyshows that the protein–detergent interactions affectthe properties of each other.DiscussionInvestigating the factors that are involved in thefibrillogenicity of α-synuclein is important to understandthe synucleinopathies, including Parkinson'sdiseases. Amphiphiles such as lipids, fatty acids anddetergents like SDS are known to affect thefibrillogenicity of the protein. Our present studyshows two important aspects in the SDS-inducedfibril formation of α-synuclein. (i) Dual behavior ofSDS in promoting fibril formation and fibril depolymerizationof α-synuclein. (ii) Our study characterizesthe fibrillogenic and non-fibrillogenicensembles of SDS–α-synuclein and shows that thefibrillogenic ensembles have highly exposed hydrophobicsurfaces and are partially structured.Figure 10 describes the dual nature of SDSinducedfibril formation and depolymerization ofα-synuclein observed in our study. Native α-synuclein (NU), though intrinsically unstructuredor natively unfolded, exhibits relatively less fibrillogenicityeven in the presence of fibril seed fornucleation. ITC, CD spectroscopy and hydrophobicdye binding along with fluorescence lifetime studiesshow that the protein–detergent ensembles (I) below2 mM SDS have extended to a partially helicalconformation with highly exposed hydrophobicsurfaces and are fibrillogenic. A recent study hasshown that, although α-synuclein lacks propersecondary structure, dynamic long-range tertiaryinteractions are present. 51 Perturbations of suchlong-range interactions by SDS may be involved inthe SDS-promoted fibril growth of α-synuclein.Our study shows that SDS inhibits fibril formationof α-synuclein at higher concentrations. The protein–detergentensembles (F) formed above 2 mMSDS, which represent fully folded α-synuclein withFigure 10. A model describing the fibrillogenic and non-fibrillogenic ensembles of α-synuclein and SDS, and theirproperties. NU represents the natively unfolded α-synuclein. I represents partially folded α-synuclein with increasedaccessible hydrophobic surfaces of the fibrillogenic ensembles. F represents the fully folded α-synuclein with lessaccessible hydrophobic surfaces of the non-fibrillogenic ensembles.

SDS-induced Fibril Growth of a-Synuclein1069less accessible hydrophobic surfaces, are less or nonfibrillogenicin nature. Such lack of fibrillogenicitymay stem either from the incompetence of theconformation for fibril growth or from the instabilityof the fibril under these conditions. Our results showthat fibril instability, which leads to dissociation ofthe fibril accompanied by conversion of a β-sheet toan α-helical conformation, is one of the factorscontributing to the non-fibrillogenic nature of α-synuclein observed above 2 mM SDS. It is importantto note that earlier studies have addressed thestructure of the protein in SDS micelles (at highconcentrations of SDS) in which α-synuclein isshown to adopt an α-helical structure. 30,45,46 Thus,these fully structured, micelle-bound species of α-synuclein are less or non-fibrillogenic in nature.α-Synuclein is known to interact with lipidmembrane. 28 The membrane environment has beenthought to be one of the factors that are involved inthe fibril formation of α-synuclein, although its exactrole remains controversial. 20,28,33–36 Our presentstudy shows a biphasic behavior in the SDS-inducedfibril growth, which parallels distinct changes in thefolded state and hydrophobic surfaces of α-synuclein.This suggests that the contradictory resultsreported with regard to the lipid vesicles could bedue to the different types of interactions of theprotein molecule with lipid vesicles under givenconditions. Such interactions can be modulated bymany factors in vivo, such as lipid composition andmembrane heterogeneity, interactions with fattyacids and cholesterol, and their role in membranepacking and fluidity alterations. Further systematicstudies are required to understand their individualroles in modulating the fibrillogenicity of α-synuclein.In this context, our present study with themembrane-mimicking agent SDS should proveuseful in understanding the role of amphiphilicmolecules in the fibrillogenicity of α-synuclein.Materials and MethodsMaterialsThioflavin T (ThT), SDS and 1,1′-bis (4-anilino) naphthalene-5,5′-disulfonicacid (bis-ANS) were purchased fromthe Sigma Chemical Company, USA. The expressionvector bearing the coding sequence of wild-type humanα-synuclein (pRK172/α-synuclein) was a kind gift fromProfessor Anthony L. Fink (University of California, SantaCruz, California).Preparation of α-synucleinWild-type α-synuclein was expressed in Escherichia coliBL21 (DE3) cells transformed with the pRK172/α-synucleinplasmid. Expression of the protein was induced bythe addition of IPTG (to 1 mM). Cells were harvested 4 hafter induction. The cell pellet was suspended in 50 mMTris–HCl (pH 7.4), 1 mM EDTA, 100 mM NaCl, 0.1 mg/mlof lysozyme and 20 μM PMSF. The suspended cells werelysed by sonication on ice and centrifuged at 5000g toremove the cell debris. The supernatant containing theexpressed α-synuclein was subjected to ammoniumsulfate fractionation. The protein that precipitated in the15%–45% saturated ammonium sulfate fraction wasresolubilized in the above-mentioned buffer, but lackinglysozyme and PMSF, and loaded onto a Sephadex G75 gelfiltrationchromatography column (150 cm×1.8 cm diameter)previously equilibrated with the same buffer. Thecolumn was eluted at a flow-rate of 20 ml/h and 2 mlfractions were collected. The fractions corresponding to α-synuclein were pooled, exchanged in 20 mM Hepes–NaOH buffer (pH 7.0), and concentrated at 4 °C using anAmicon ultrafiltration unit with a 5 kDa cutoff filter. Theprotein preparation was found to be highly homogeneousas assessed by SDS-PAGE (15% polyacrylamide gel). Theextinction coefficient of α-synuclein, determined asdescribed, 52 was 0.354 at 280 nm for a 1 mg/ml solution.The concentration of α-synuclein was determined usingthis extinction coefficient.Fibril growth of α-synucleinPurified α-synuclein was filtered through 0.22 μm poresize filters to remove any particulate matter or biggeraggregates. The fibrils of α-synuclein were obtainedinitially by incubating α-synuclein (1 mg/ml) in 20 mMHepes–NaOH buffer (pH 7.0), containing 0.15 mM SDS at37 °C overnight with vigorous stirring. Formation of thefibrils was measured by ThT binding: 53 at various timepoints,a small portion of the sample was added to 1 mlof 10 μM ThT in 50 mM glycine–NaOH buffer (pH 8.5),and the fluorescence intensity at 485 nm upon excitingthe sample at 445 nm was measured using a Hitachi F-4000 fluorescence spectrophotometer. The fluorescenceintensity is proportional to the extent of fibril formation.The fibril sample was used for the preparation offragmented fibril seeds by sonication. In all furtherexperiments, seeded fibril growth of α-synuclein wasstudied using these fibril seeds. No stirring was used inthese experiments.Seeded fibril growth of α-synuclein was studied byincubating the sample of α-synuclein (at the indicatedconcentrations) in 20 mM Hepes buffer (pH 7.0), in theabsence or in the presence of indicated concentrations ofSDS with 10% fibril seeds (with respect to the monomericα-synuclein concentration) at 37 °C. A 10 μl portion of thesample was withdrawn at different time-points andadded to 1 ml of 10 μM ThT in 50 mM glycine–NaOHbuffer (pH 8.5), and the ThT fluorescence was measuredas described above.Electron microscopyA portion of the sample of α-synuclein fibrils was placedon Formavar/carbon-coated grids (300-mesh). Excesssample was removed and the grid was air-dried. Thefibril-bearing grid was stained with 2% (w/v) uranyl acetatefor 2 min. Images from randomly selected areas werecaptured on a film at 33,000–50,000× magnification. Imageswere processed and printed using Adobe Photoshop.Isothermal titration calorimetryITC of α-synuclein in the presence of SDS wasperformed using a VP-ITC instrument (Microcal, Northampton,MA 01060, USA). In all titration experiments,7 mM SDS in 20 mM Hepes–NaOH buffer (pH 7.0) wasinjected (10 μl per addition) into the cell containing the

1070 SDS-induced Fibril Growth of a-Synucleingiven concentration of α-synuclein in the same buffer at30 °C. In a control experiment (blank titration), thecorresponding SDS solution was injected into the bufferwithout the protein.bis-ANS bindingThe sample of α-synuclein (1 mg/ml or 0.22 mg/ml) in20 mM Hepes–NaOH buffer (pH 7.0) containing 5 μM bis-ANS was titrated with increasing concentrations of SDS.The fluorescence spectrum of bis-ANS was recorded usinga Hitachi F-4000 fluorescence spectrophotometer with theexcitation wavelength set at 390 nm. The excitation andemission band passes were set at 5 nm. All the spectrawere recorded in the corrected spectrum mode.Time-resolved fluorescence measurementsFluorescence lifetime measurements were performedusing the TCSPC Picosecond Lifetime measurementsystem, model 5000 U (Horiba Jobin Yvon, France) intime-correlated single photon counting (TCSPC) mode.The excitation source was a 337 nm wavelength NanoLEDpulsed laser with a repetition rate of 250 kHz. Theinstrument response function (prompt) was obtained at337 nm using Ludox suspension. The sample of α-synuclein (0.22 mg/ml) in 20 mM Hepes–NaOH buffer(pH 7.0) containing 5 μM bis-ANS was titrated withincreasing concentrations of SDS. The emission decay (at485 nm) data were analyzed using the DAS6softwareprovided with the instrument. The analysis used astatistical method of iterative non-linear, least-squaresreconvolution, assuming the three-exponential decayfunction:ðA þ B 1 exp½ i=s 1 ŠþB 2 exp½ i=s 2 ŠþB 3 exp½ i=s 3 ŠÞThe best fit was assessed based on χ2, which was closeto 1 for all the samples. The goodness of the fit is evidentalso from the distribution of weighted residuals along thezero line. In another experiment, a sample of SDS micelle(20 mM) in the same buffer containing 5 μM bis-ANS wasused. The decay data could be fit best with a twoexponentialdecay model.Circular dichroism spectroscopyFar-UV CD spectra (200–250 nm) of α-synuclein (eitherat 1 mg/ml or 0.22 mg/ml) in 20 mM Hepes–NaOH buffer(pH 7.0) in the absence or in the presence of variousconcentrations of SDS were recorded at room temperatureusing a Jasco J-715 spectropolarimeter. The spectra shownare the average of four scans.SDS concentration-dependent dissociation ofα-synuclein fibrilsFibrils of α-synuclein (1 mg/ml with respect to theconcentration of monomeric protein) were prepared asdescribed earlier. The fibrils were resuspended at a finalconcentration of 0.2 mg/ml in 20 mM Hepes buffer (pH7.0) in the presence of various concentrations of SDS andthe samples were incubated at 37 °C without agitation. A(5 μl) portion was withdrawn from the samples atdifferent time-points and added to 1 ml of 10 μM ThT in50 mM glycine–NaOH buffer (pH 8). The fluorescence offibril-bound ThT, which is a measure of amount of fibrilspresent in the sample at the indicated time-point, wasmeasured as described earlier.Far-UV CD spectral changes upon fibril dissociationwere studied by incubating the α-synuclein fibrils (1 mg/ml) in 20 mM Hepes (pH 7.0) in the presence of either0.5 mM or 4 mM SDS at 37 °C. The CD spectra wererecorded at the indicated incubation time-point using aJasco J-715 spectropolarimeter. Spectra shown are theaverage of four scans.Light-scattering of α-synuclein upon treating withSDSTo test whether treating α-synuclein with SDS promotesamorphous aggregation, a sample of 0.3 mg/ml of α-synuclein in 20 mM Hepes–NaOH buffer (pH 7.0) wasequilibrated at 37 °C in the cuvette holder of a Hitachi F-4000 fluorescence spectrophotometer and the sample wastreated with increasing concentration of SDS. Lightscatteringwas measured by setting both the excitationand emission monochromator at 465 nm. Similarly, asample of 0.3 mg/ml of human recombinant αB-crystallinin the same buffer was treated with SDS and the lightscatteringwas measured.Measurement of critical micellar concentration (cmc)of SDSThe cmc of SDS in 20 mM Hepes–NaOH buffer (pH 7.0)was measured using the fluorescent probe diphenylhexatriene.50 The fluorescence intensity of the probe(10 μM) at 430 nm was measured in buffer alone or in thepresence of 0.3 mg/ml of α-synuclein with differentconcentrations of SDS using a Hitachi F-4000 fluorescencespectrophotometer. The probe was excited at 358 nm. Theexcitation and emission band passes were set at 1.5 nm and10 nm, respectively.AcknowledgementsWe thank Professor Antony L. Fink (University ofCalifornia, California, USA) for the kind gift ofpRK172-α-synuclein plasmid and Dr Shashi Singh(Centre for Cellular and Molecular Biology, Hyderabad,India) for electron microscopy. 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