Supplementary material for
Structural characterization of alpha-synuclein in an aggregation prone
state
Min-Kyu Cho,1 Gabrielle Nodet,3 Hai-Young Kim,1 Malene R Jensen,3 Pau Bernado,5
Claudio O Fernandez,4 Stefan Becker,1 Martin Blackledge,3 Markus Zweckstetter1, 2, *
1
Department for NMR-based Structural Biology, Max Planck Institute for Biophysical
Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
2
DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen,
Germany
3
Institut de Biologie Structurale Jean-Pierre Ebel, CEA-CNRS-UJF UMR 5075, 41 Rue
Jules Horowitz, Grenoble 38027, France
4
Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de
Investigaciones Científicas y Técnicas, Universidad Nacional de Rosario, Suipacha 531,
S2002LRK Rosario, Argentina
5
Institute for Research in Biomedicine, Parc Científic de Barcelona, 08028 Barcelona,
Spain
*
Corresponding author. Department for NMR-based Structural Biology, Max Planck
Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany. Fax:
49 551 201 2220. E-mail address: mzwecks@gwdg.de
1
Supplementary Fig. 1. Differences in experimental C chemical shifts observed at pH
3.0 and pH 7.4 as a function of residue number. The largest differences were observed in
proximity to Asp and Glu residues. The C difference values are directly calculated form
the experimental values without the use of random coil values.
2
V125
V37
V26
Supplementary Fig. 2. Superposition of a selected portion of the 1H-15N HSQC spectra
of A18C S in the diamagnetic (blue) and paramagnetic state (red). The cross peak of
V26 disappears in the paragmagnetic state. The intensity of the cross peak of V37
decreases, while that of V125 shows a comparable intensity in both conditions.
3
Supplementary materials and methods
1.1. Protein Preparation
pT7-7 plasmid encoding for human wt S was kindly provided by the Lansbury
Laboratory, Harvard Medical School, Cambridge, MA. Plasmids containing αS variants
were expressed in Escherichia coli BL21 (DE3) cells. Protein expression and purification
was performed as previously described with minor changes.1 For production of
labeled proteins, M9-minimal medium supplemented with
Laboratories) was used. For
13
C/15N-labeled samples,
13
15
15
N-
NH4Cl (Cambridge Isotope
C-Glucose (Cambridge Isotope
Laboratories) was also added to the M9-minimal medium.
Standard solid-phase peptide synthesis was used to produce a peptide comprising
residues 105 to 136 of αS. The peptide was purified by reverse phase HPLC and the
purity (>95%) was analyzed by mass spectrometry. NMR samples contained ~ 0.1 mM
15
N- or 15N /13C-labelled S in 20 mM Na acetate, 100 mM NaCl, pH 3.0.
1.2. Spin Labeling of S
The nitroxide spin label chosen for reaction with the cysteine-containing mutants
was
MTSL
(1-oxy-2,2,5,5-tetramethyl-D-pyrroline-3-methyl)-methanethiosulfonate
(Toronto Research Chemicals, Toronto, Ontario, Canada). MTSL had already proven to
efficiently react with S cysteine mutants, and the reaction was carried out as described
previously.2
1.3. Alignment of S in Anisotropic Media
4
Residual dipolar couplings (RDCs) were measured for S aligned in 5% w/v noctyl-penta(ethylene glycol)/octanol (C8E5) (Sigma).3 Formation of the anisotropic, dilute
liquid crystalline phase was monitored by the splitting of the deuterium signal, which was
18 ± 2 Hz at pH 7.4 and 22.5 ± 2 Hz at pH 3.0.
1.4. NMR Spectroscopy
The experiments were recorded on Bruker Avance 600, 700 and 800 MHz NMR
spectrometers at 15 C. Data processing was performed using the software packages
NMRPipe/NMRDraw,4 Topspin (Bruker) and Sparky.5 A pH titration using twodimensional (2D) 1H-15N Heteronuclear Single Quantum Coherene (HSQC) spectra in
combination with three-dimensional triple-resonance experiments (HNCO, HACANNH,
HNCACB) was performed, to obtain sequence-specific assignments for the backbone of
of S at pH 3.0. Secondary shift values were analyzed using the SSP program.6
Pulsed-field gradient NMR experiments were acquired at 15 °C on unlabeled S
(100 M) dissolved in 99.9 % D2O, 100 mM NaCl and containing dioxane (~20 mM) as
an internal radius standard and viscosity probe.7 Sixteen one-dimensional 1H spectra were
collected as a function of gradient strength varying between 2 % and 95 % of its
maximum value. After baseline correction, the decay in the intensity of the signals from
the aliphatic region was fitted as a function of gradient strength (g) to the equation f(g) =
Ae-d(g^2).
J(HNH) scalar couplings were measured using a 2D intensity-modulated 1H-15N
3
HSQC spectrum (48 scans, relaxation delay 1.2 ms, 2τ = time for evolution of 3JHNHα: 30
5
ms).8 Coupling values were calculated from the intensity ratios using the relation
Scross/Sdiag = cos(π3JHNHα2τ).
One-bond N-H RDCs (DNH) were acquired using the 2D inphase-antiphase HSQC
sequence9 under both isotropic and anisotropic conditions. DNH values were calculated as
the difference between splittings measured in an aligned sample and those measured in
the isotropic phase (RDCs were not corrected for the negative gyromagnetic ratio of 15N).
Paramagnetic relaxation enhancement (PRE) effects were measured from the peak
intensity ratios between two 2D 15N-1H HSQC spectra with a paramagnetic sample and a
diamagnetic sample, respectively. Paramagnetic samples were measured at 100 M
MTSL-labeled protein concentration, and diamagnetic experiments were performed with
100 M protein in the presence of 0.5 mM ascorbic acid (Sigma), which reduces MTSL
at acidic pH.
15
N longitudinal relaxation rate in rotating frame (R1) and transverse relaxation
rate (R2) as well as
15
N heteronuclear NOEs were measured at 600 MHz proton
frequency at 288 K. For the heteronuclear NOE experiment, 1H saturation was achieved
by the application of 120 1H pulses separated by 5 ms, for 3 s period. Eight time points
were collected for both R2 and R1 experiments, and each set included duplicate
measurements for estimation of the uncertainty. The delays used were 7.6, 30.4, 76.0,
152.0, 243.2, 304.0, 380.0 and 547.2 ms for R2and 8.0, 48.0, 80.0, 160.0, 240.0, 320.0,
400.0, and 480.0 for R1experiment. Time points that were measured twice are shown in
italic.
1.5. Flexible Meccano - Interpetation of Ensemble-Averaged Relaxation Rates
6
Flexible-meccano, an algorithm developed to sample the conformational space
available to disordered proteins in terms of explicit ensembles of molecules was used.
Unbiased conformational ensembles of 10000 structures were calculated, and effective
relaxation rates for each conformer calculated in the presence of the four spin probes.
Relaxation rates for each conformer are described using the modelfree formalism
introduced for interpretation of 1H-1H dipole-dipole cross relaxation (nOe) interactions,10
and recently adapted to the calculation of paramagnetic relaxation enhancement by Clore
et al,11 Radial and angular order parameters were calculated over the positions of all
allowed spin-probes. Rotameric sampling propensities derived from ESR and molecular
dynamics simulation were used to describe possible positions of the MTSL probe 12. The
correlation time for the electron-nuclear interaction τc was set to 4 ns, in agreement with
previous studies.13 The average relaxation rate was calculated over all individual
backbone confomers. Experimental values for the intrinsic transverse relaxation rates14
were used to account for the diamagnetic contribution to the spin relaxation rate.
Sub-ensembles were then selected on the basis of agreement with respect to the
experimental data using a genetic algorithm. Simulations indicated that for a molecule of
this size with four spin labels, ensembles of 80 structures presented a reasonable
description of the conformational behaviour while avoiding over-fitting. A more detailed
description of this algorithm is in preparation. The identical procedure was applied for
both samples, and contact maps showing a logarithmic comparison between the selected
ensemble and the original unbiased ensemble are used to compare the data.
For the ensembles of 80 conformations the hydrodynamic radius of each
conformation was computed with a modified version of the program HydroPRO15 using
7
an atomic element radius of 3.3 Å to define the volume of the conformation. The bead
modelling strategy adopted by HydroPRO has been proven to be efficient to calculate
hydrodynamic properties for both rigid and flexible molecules.15, 16 The averaged Rh was
obtained as follows:
1
1
1


Rh N i 1,N Rh,i
(1)
, where N is the number of conformations of the ensemble and Rh,i is the hydrodynamic

radius of the conformation i.
8
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