A R T I C L E
Solid-state NMR sequential assignments of
a-synuclein
Julia Gath•Birgit Habenstein•Luc Bousset•
Ronald Melki•Beat H. Meier•Anja Bo¨ckmann
Received: 3 March 2011 / Accepted: 27 June 2011 / Published online: 9 July 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Parkinson’s disease is amongst the most fre-quent and most devastating neurodegenerative diseases. It is tightly associated with the assembly of proteins into high-molecular weight protein species, which propagate between neurons in the central nervous system. The prin-cipal protein involved in this process is a-synuclein which is a structural component of the Lewy bodies observed in diseased brain. We here present the solid-state NMR sequential assignments of a new fibrillar form of this pro-tein, the first one with a well-ordered and rigid N-terminal part.
Keywords a-Synuclein Fibrils Solid-state NMR Assignments Secondary structure
Biological context
a-Synuclein is one of the major proteins involved in Par-kinson’s disease. Aggregates of this normally soluble protein are found in Lewy Bodies which are associated to this disability (Brundin et al. 2010; Eller and Williams
2011). The superstructures of these aggregates, as seen by electron microcopy, are protein fibrils; the aggregation mechanism of a-synuclein, as well as the structure of the resulting fibrils, remain however unknown. Structural studies of a-synuclein fibrillar assemblies are challenging today as neither of the classical methods which allow to study protein structures at a molecular level, as X-ray crystallography or NMR in solution, are adequate tools for this task. Solid-state NMR has recently developed to become a powerful tool to reveal conformational details and atomic-resolution structures of insoluble and non-crystalline molecular assemblies (Van Melckebeke et al.
2010; Bo¨ckmann and Meier 2010; Renault et al. 2010; Debelouchina et al.2010; Jehle et al.2010; Wasmer et al.
2008). Initial structural studies by solid-state NMR of a-synuclein fibrils, or fragments thereof, have been per-formed by several groups (Heise et al. 2005, 2008; Kloepper et al.2006,2007; Vilar et al.2008; Loquet et al.
2010) and 48 (form A) and 23 (form B) residues were sequentially assigned in ref (Heise et al.2005) and 26 in ref (Kloepper et al. 2007). A comparison of the published spectra reveals the appearance of different polymorphs, as well as significant heterogeneity, in some individual sam-ples indicative of differences in the conformation and packing of a-synuclein molecules within or between the different fibrils studied. Sample preparation is a crucial step in fibril structural studies, as structural differences or polymorphs can arise from different production, purifica-tion and assembly condipurifica-tions. Extreme care has to be
Julia Gath, Birgit Habenstein and Luc Bousset have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s12104-011-9324-3) contains supplementary material, which is available to authorized users.
J. Gath B. H. Meier (&)
Physical Chemistry, ETH Zu¨rich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland
e-mail: beme@ethz.ch
B. Habenstein A. Bo¨ckmann (&)
Institut de Biologie et Chimie des Prote´ines, UMR 5086 CNRS/ Universite´ de Lyon 1, 7 passage du Vercors, 69367 Lyon, France e-mail: a.bockmann@ibcp.fr
L. Bousset R. Melki (&)
Laboratoire d’Enzymologie et Biochimie Structurales, UPR 3082 CNRS, Avenue de la Terrasse,
91198 Gif-sur-Yvette, France e-mail: melki@lebs.cnrs-gif.fr DOI 10.1007/s12104-011-9324-3
exercised when comparing results from samples of differ-ent sources or even differdiffer-ent batches. We here presdiffer-ent the sequential resonance assignments of an a-synuclein poly-morph for which for the first time the N-terminal residues, postulated not to be part of the core in other studies (Vilar et al.2008; Miake et al.2002), are assigned and shown to be mainly in b-sheet conformation. This polymorph might show similarities to the B-form described by Heise et al. (2005) since for 8 residues the chemical shift assignments coincide with the here presented ones. The spectra also overlay well with those published for a yet unassigned polymorph (Loquet et al.2010).
Methods and experiments Protein expression and purification
Full-length a-synuclein was expressed in E. coli BL21 DE3 codon ? cells (Stratagene). Cells were grown in LB medium to an Abs600nmof 0.8 and sedimented at 3,000g for
10 min in 1 l tubes. The pelleted bacteria were washed with 200 ml of M9 salt and spun at 3,000g for 10 min. The bacterial pellets were resuspended in half the volume where they originally grew of M9 media containing 1.75 g of 15NH4Cl, 2.5 g of U-13C glucose, 2 mM MgSO4,
0.1 mM CaCl2, 10 lg thiamine per litre of culture. Cells
were grown for 30 min at 37°C and a-synuclein expression was induced by 0.5 mM IPTG for 3 h. The cells were then harvested by centrifugation (4,000g, 10 min), resupended into lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 1 mM PMSF) and lysed by sonication. Cell extracts were clarified by centrifugation at 14,000g, 30 min. Purification was performed as previously described (Hansen et al.2011). Sample preparation
a-Synuclein was dialyzed 16 h against 5 mM Tris–HCl pH 7.5 in double-distilled H2O at 8°C. Fibrillation was
achieved at 300 lM by shaking 1 ml solution aliquots at 37°C in an Eppendorf Thermomixer set at 600 rpm with a 3 mm orbital for 7 days. Fibrils were spun for 20 min at 20° C, 50,000 rpm on a TL100 ultracentrifuge (Beckman) using the TLA 100.4 rotor. The pelleted material was used to fill a ZrO2 3.2 mm rotor (Bruker) using a home made
filling device (Bo¨ckmann et al.2009) spun at 40,000 rpm for 16 h at 18°C in an SW60 TI rotor and an optima L90-K ultracentrifuge (Beckman).
NMR spectroscopy
NCOCA, CANCO, NCACO and CCC experiments to perform the assignment as described in detail in references (Habenstein et al., accepted, Schuetz et al. 2010) for sequential assignments. The sequential walk was achieved by connecting resonances from NCACO/NCACB, CANCO/CAN(CO)CA and NCOCA spectra. Sequential connections were also present in CCC spectra. Side-chain assignments were done using NCACB and CCC spectra. Full experimental details are given in Table S1. All assignment spectra were recorded on the same rotor. However, 2D DARR spectra were taken of four different preparations to check reproducibility of the sample prepa-ration. All preparations yielded identical spectra.
Assignment and data deposition
The a-synuclein sample studied here reveals a well-resolved 2D13C–13C spectrum with narrow lines, shown in Fig.1 (extract of the aliphatic and carbonyl regions). Assignments are given for crosspeaks corresponding to directly bonded carbons (in the aliphatic region only below the diagonal). A representative plane of the 3D NCACB, NCACO, CANCO and NCOCA experiments is shown in Fig.2. The observed line width in the spectra is mostly comprised between 0.5 and 1 ppm. The lines are not quite as narrow as in HET-s(218–289) (Siemer et al. 2005), but allow for an assignment using the afore mentioned 3D methods. Sequential assignments could be achieved for 85% of residues 1–97 and for 74% of all carbon atoms and 82% backbone amide nitrogen atoms. The chemical shifts have been deposited in the BMRB under the accession number 17498. The C-terminal portion including residues 98–140 seems to be flexible, as no signals could be attributed to this region. However, all amino-acid types present in the C-terminal region could be observed in the scalar-coupling based 1H–13C INEPT spectrum (see Sup-plementary Figure S1). For the stretch ranging from residue 44–57, unambiguous sequential assignments were difficult due to lower signal/noise or spectral overlap. Figure S2 compares the intensities of resonances of some assigned residues with ‘‘weak’’ peaks tentatively assigned to resi-dues in this stretch. Tentative assignments are not consid-ered in the following and were not submitted to the BMRB. Assigned atoms are shown in the assignment graph in Figure S3. Most resolved and strong cross peaks in a 20 ms 2D DARR spectrum can be explained based on this assignment. Most weaker peaks in this spectrum are due to sequential contacts and can also be explained by the assignments.
13C chemical shift / ppm 13C chemical shift / ppm 32K 33T 9S 73G 35E 90A 17A 11A 83E 31G 72T 38L 93G 42S 81T 61E 36G 64T 10K 3V 82V 75T 27A 63V 95V 61E 78A 5M 65N 8L 80K 66V 14G 43K 12K 69A 21K 84G 67G 26V 54T 30A 40V 24Q 24Q 60K 96K 2D 76A 58K 79Q 47G 87S 34K 62Q 20E 35E 16V 91A 19A 97K 28E 15V 37V 70V 74V 77V 1M 48V 2D 6K 22T 59T 20E 53A 23K 79Q 83E 71V 18A 39Y 89A 86G 55V 7G 13E 41G 29A 25G 51G 92T 28E 88I 85A 68G 13E 60K 80K 70V 15V 37V 1M 1M 32K 23K 23K 64T 28E 28E 22T 18A 59T 71V 23K 96K 2D 95V 30A 61E 61E 81T 58K 16V 38L 88I 75T 40V 75T 60K 8L 88I 55V 62Q 77V 42S 91A 16V 88I 10K 16V 3V 9S 77V 13E 13E 79Q 97K 97K 81T 21K 21K 6K 69A 40V 26V 78A 92T 72T 8L 55V 15V 49V 70V 63V 82V 22T 19A 74V 8L 8L 76A 26V 90A 48V 54T 40V 97K 71V 5M 88I 15V 27A 70V 3V 43K 35E 35E 29A 53A 39Y 79Q 37V 87S 59T 64T 55V 85A 11A 24Q 24Q 52V 95V 33T 38L 23K 63V 6K 6K 77V 20E 20E 92T 26V 37V 97K 34K 95V 17A 21K 38L 96K 82V 83E 83E 48V 80K 6K 65N 21K 89A 5M 74V 12K 72T 38L 32K 33T 74V 170 180 190 70 60 50 40 30 20 20 30 40 50 60 70 Fig. 1 2D13C–13C DARR Spectrum of a-synuclein fibrils recorded with a mixing time of 20 ms at a magnetic field of 14.1 Tesla. The data was zero-filled and apodized in both dimensions using a 0.3 shifted squared sine bell function. The spectrum was processed using NMRPipe (Delaglio et al.
1995). All one-bond
correlations are labeled (in the aliphatic region only below the diagonal) 24QCb,C,25GN 24QCg,C,25GN 25GCa 24QCa,C,25GN 25GCa,C,26VN 78ACa,C,79QN 78ACb,C,79QN 30 40 50 26ValC,Ca,N 25GC,Ca,N 9SC,Ca,N 32K’C,Ca,N 36GC,Ca,N 79QC,Ca,N 42SC,Ca,N 59TC,Ca,N 24EC, 25GN,Ca 35EC, 36GN,Ca 78AC, 79QN,Ca 41GC, 42SN,Ca 31GC, 32KN,Ca 8LC, 9SN,Ca 58KC, 59TC,N 25GC, N, 26VCa 14GC, 15VC,Ca 50 60 δ2/1 ( 13 C) / ppm δ3(13C) / ppm δ1/2(15N) = 114.4 ppm 25GC, 24QC, 25GN 24QC, 25GC, 26VN 26VC, 25GC, 26VN 160 170 180 170 180 NCACB NCACO CANCO NCACO NCOCA NCOCA 25GCa,Ca,N 79QCg,Ca,N 32KCb,Ca,N 26VCb,Ca,N 26VCgb,Ca,N 26VCga,Ca,N 36GCa,Ca,N
Fig. 2 Planes of 3D NCACO, CANCO, NCOCA and NCACB spectra. For experimental details see Table S1. The data were analyzed using the CCPN software (Fogh et al.2002). Assignments of
the peaks are given. Grey labels mean that the maximum of the corresponding peak is at a different N-shift, therefore leading to weaker signals in the plane presented
conformation. The assigned regions do not correspond to the regions previously assigned by (Heise et al. 2005,
2008) for both A- and B-forms, who found the first 37 N-terminal residues statically disordered. Here, we observe most of the N-terminal residues of the protein up to residue 97, with an exception being residues 44–57, which are potentially less structurally homogeneous or more mobile. The structured regions observed in our sample correspond however to the regions identified in the presence of detergent to be a-helical (Bussell and Eliezer2003). In the moment we prefer not to add another structural model to the flurry of already existing ones, but note that the sequential assignment of a-synuclein presents a first step towards a high-resolution structure of this protein, which seems perfectly feasible.
Acknowledgments This work was supported by the Agence Na-tionale de la Recherche (ANR-07-PCVI-0013-03, ANR-PCV08 321323 and ANR08-PCVI-0022-02), the ETH Zurich, the Swiss National Science Foundation (Grant 200020_124611), the Era-Net Neuron (project MIPROTRAN, ANR-08-NEUR-001-01) and the Centre National de la Recherche Scientifique. We also acknowledge support from the European Commission under the Seventh Frame-work Programme (FP7), contract Bio-NMR 261863.
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