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

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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.
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