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

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. M.F.Aacknowledges the Council of Scientific and IndustrialResearch, New Delhi, India for the grant of aSenior Research Fellowship.References1. Forster, E. & Lewy, F. H. (1912). Paralysis agitans. InPathologische Anatomie. Handbuch der Neurologie(Lewandowski, M., ed), pp. 920–933, Springer Verlag,Berlin.2. Forno, L. S. (1996). Neuropathology of Parkinson'sdisease. J. Neuropathol. Expt. Neurol. 55, 259–272.3. Spillantini, M. G., Crowther, R. A., Jakes, R.,Hasegawa, M. & Goedert, M. (1998). Alpha-synucleinin filamentous inclusions of Lewy bodies fromParkinson's disease and dementia with lewy bodies.Proc. Natl Acad. Sci. USA, 95, 6469–6473.