Abstract

Article10 September 2009free access Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models Damla Pinar Karpinar Damla Pinar Karpinar Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany International Max Planck Research School for Molecular Biology, Göttingen, Germany Search for more papers by this author Madhu Babu Gajula Balija Madhu Babu Gajula Balija Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany International Max Planck Research School for Molecular Biology, Göttingen, Germany Search for more papers by this author Sebastian Kügler Sebastian Kügler DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department of Neurology, Medical School, University of Göttingen, Göttingen, Germany Search for more papers by this author Felipe Opazo Felipe Opazo DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Nasrollah Rezaei-Ghaleh Nasrollah Rezaei-Ghaleh Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Nora Wender Nora Wender Molecular Neurogenetics Group, European Neuroscience Institute, Göttingen, Germany Search for more papers by this author Hai-Young Kim Hai-Young Kim Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Grit Taschenberger Grit Taschenberger Department of Neurology, Medical School, University of Göttingen, Göttingen, Germany Search for more papers by this author Björn H Falkenburger Björn H Falkenburger DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Henrike Heise Henrike Heise Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Ashutosh Kumar Ashutosh Kumar Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Dietmar Riedel Dietmar Riedel Department for Electron Microscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Lars Fichtner Lars Fichtner DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University of Göttingen, Göttingen, Germany Search for more papers by this author Aaron Voigt Aaron Voigt DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Gerhard H Braus Gerhard H Braus DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University of Göttingen, Göttingen, Germany Search for more papers by this author Karin Giller Karin Giller Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Stefan Becker Stefan Becker Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Alf Herzig Alf Herzig Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Marc Baldus Marc Baldus Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Herbert Jäckle Herbert Jäckle Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Stefan Eimer Corresponding Author Stefan Eimer DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Molecular Neurogenetics Group, European Neuroscience Institute, Göttingen, Germany Search for more papers by this author Jörg B Schulz Corresponding Author Jörg B Schulz DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Christian Griesinger Corresponding Author Christian Griesinger Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Search for more papers by this author Markus Zweckstetter Corresponding Author Markus Zweckstetter Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Search for more papers by this author Damla Pinar Karpinar Damla Pinar Karpinar Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany International Max Planck Research School for Molecular Biology, Göttingen, Germany Search for more papers by this author Madhu Babu Gajula Balija Madhu Babu Gajula Balija Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany International Max Planck Research School for Molecular Biology, Göttingen, Germany Search for more papers by this author Sebastian Kügler Sebastian Kügler DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department of Neurology, Medical School, University of Göttingen, Göttingen, Germany Search for more papers by this author Felipe Opazo Felipe Opazo DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Nasrollah Rezaei-Ghaleh Nasrollah Rezaei-Ghaleh Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Nora Wender Nora Wender Molecular Neurogenetics Group, European Neuroscience Institute, Göttingen, Germany Search for more papers by this author Hai-Young Kim Hai-Young Kim Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Grit Taschenberger Grit Taschenberger Department of Neurology, Medical School, University of Göttingen, Göttingen, Germany Search for more papers by this author Björn H Falkenburger Björn H Falkenburger DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Henrike Heise Henrike Heise Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Ashutosh Kumar Ashutosh Kumar Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Dietmar Riedel Dietmar Riedel Department for Electron Microscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Lars Fichtner Lars Fichtner DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University of Göttingen, Göttingen, Germany Search for more papers by this author Aaron Voigt Aaron Voigt DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Gerhard H Braus Gerhard H Braus DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University of Göttingen, Göttingen, Germany Search for more papers by this author Karin Giller Karin Giller Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Stefan Becker Stefan Becker Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Alf Herzig Alf Herzig Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Marc Baldus Marc Baldus Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Herbert Jäckle Herbert Jäckle Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany Search for more papers by this author Stefan Eimer Corresponding Author Stefan Eimer DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Molecular Neurogenetics Group, European Neuroscience Institute, Göttingen, Germany Search for more papers by this author Jörg B Schulz Corresponding Author Jörg B Schulz DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany Search for more papers by this author Christian Griesinger Corresponding Author Christian Griesinger Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Search for more papers by this author Markus Zweckstetter Corresponding Author Markus Zweckstetter Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany Search for more papers by this author Author Information Damla Pinar Karpinar1,2,3,‡, Madhu Babu Gajula Balija1,2,3,‡, Sebastian Kügler2,4,‡, Felipe Opazo2,5,‡, Nasrollah Rezaei-Ghaleh1, Nora Wender6, Hai-Young Kim1, Grit Taschenberger4, Björn H Falkenburger2,5, Henrike Heise1, Ashutosh Kumar1, Dietmar Riedel7, Lars Fichtner2,8, Aaron Voigt2,5, Gerhard H Braus2,8, Karin Giller1, Stefan Becker1, Alf Herzig9, Marc Baldus1, Herbert Jäckle9, Stefan Eimer 2,6, Jörg B Schulz 2,5, Christian Griesinger 1,2 and Markus Zweckstetter 1,2 1Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany 2DFG Research Center for the Molecular Physiology of the Brain (CMPB), Göttingen, Germany 3International Max Planck Research School for Molecular Biology, Göttingen, Germany 4Department of Neurology, Medical School, University of Göttingen, Göttingen, Germany 5Department for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, Göttingen, Germany 6Molecular Neurogenetics Group, European Neuroscience Institute, Göttingen, Germany 7Department for Electron Microscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany 8Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University of Göttingen, Göttingen, Germany 9Department of Molecular Developmental Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, Göttingen, Germany ‡These authors contributed equally to this work *Corresponding authors: Department for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany. Tel.: +49 551 201 2220; Fax: +49 551 201 2202; E-mail: [email protected] for NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen 37077, Germany. Tel.: +49 551 201 2201; E-mail: [email protected] for Neurodegeneration and Restorative Research, Center for Neurological Medicine, University of Göttingen, 37073 Göttingen, Germany. Tel.: +49 551 39 13540; Fax: +49 551 39 13541; E-mail: [email protected] Neurogenetics Group, European Neuroscience Institute, 37077 Göttingen, Germany. Tel.: +49 551 12379; Fax: +49 551 39 10129; E-mail: [email protected] The EMBO Journal (2009)28:3256-3268https://doi.org/10.1038/emboj.2009.257 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The relation of α-synuclein (αS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure-based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function. Introduction The pathological hallmark of Parkinson's disease (PD) and several other neurodegenerative disorders is the deposition of intracytoplasmic neuronal inclusions termed Lewy bodies (Goedert, 2001). The major components of Lewy bodies are amyloid fibrils of the protein α-synuclein (αS) (Spillantini et al, 1997). Mutations of αS associated with familial PD (A30P, A53T, E46K) have an increased aggregation propensity in vitro, in agreement with the aggregation of αS into fibrillar Lewy bodies in vivo (Conway et al, 2000; Greenbaum et al, 2005). To study the pathogenic events of PD, a wide range of model systems have thus been described that rely on overexpression of αS and range in complexity from in vitro, to yeast, cell culture, worm, fly, mouse and primate (Feany and Bender, 2000; Nass and Blakely, 2003; Outeiro and Lindquist, 2003; Meredith et al, 2008). In several of these model systems, the rate of fibril and inclusion body formation does not correlate with neurotoxicity (Outeiro and Lindquist, 2003; Chen and Feany, 2005; Volles and Lansbury, 2007). This lack of correlation forms the basis for the hypothesis that small oligomers, but not fibrils, are the most toxic species of αS (Lashuel and Lansbury, 2006). However, the functions of different aggregated αS species for neurotoxicity in vivo are still unclear (Cookson and van der Brug, 2008), as overexpression of wild type, genetic mutants, phosphorylation mimics and truncation mutants of αS result in various aggregate species in vivo and induce different degrees of toxicity in different PD model systems (Feany and Bender, 2000; Masliah et al, 2000; Outeiro and Lindquist, 2003; Chen and Feany, 2005; Periquet et al, 2007; Volles and Lansbury, 2007; Gorbatyuk et al, 2008). Moreover, structural data for aggregation intermediates of αS are highly limited because of their transient and heterogeneous nature (Lashuel and Lansbury, 2006). To obtain insight into the structure of different aggregate species of αS and their toxicity in vivo we designed, based on the structural information of the monomeric and fibrillar state of αS, αS mutants with radically different aggregation properties. We then correlated their ability to form amyloid fibrils with the degree of neurotoxictiy in four established model systems for PD (human embryonic kidney cells (Opazo et al, 2008), C. elegans (Nass and Blakely, 2003), Drosophila (Feany and Bender, 2000) and mammalian neurons (Zhou et al, 2000)). The structural properties of the aggregates formed by the αS mutants were characterized at single-residue resolution by solid-state nuclear magnetic resonance (NMR) spectroscopy. Results Structure-based design mutants of αS delay fibril formation We based our design on the conformational properties of the αS monomer in solution and on the topology of αS fibrils known from previous NMR measurements (Bertoncini et al, 2005; Heise et al, 2005; Vilar et al, 2008). The genetic mutation A30P is located in a region of αS that is statically disordered in amyloid fibrils (Heise et al, 2005). To interfere with aggregation, we moved the single proline mutation found in the genetic A30P mutant to a position that is part of the β-sheet rich core of αS fibrils (Figure 1A). We selected Ala 56 and Ala 76 that are characterized by relatively large residual dipolar coupling values in the soluble monomer, suggestive of a rigid nature (Bertoncini et al, 2005). The following αS variants were generated: A30P, A56P, A76P, A30P/A56P, A30P/A76P and A30P/A56P/A76P (TP αS). Figure 1.Structure-based design mutants impair fibrillation of αS. (A) Functional domains of αS. Familial mutants (black) and structure-based design mutants (red) are labelled along the sequence. Regions involved in β-sheet formation in the fibril (Heise et al, 2005) are marked (purple). (B) Fibril formation of wt αS (black), A56P αS (yellow) and TP αS (red) followed by Thioflavin T fluorescence (ThT). Data for A30P, A76P, A30PA56P, A30PA76P are shown in Supplementary Figure S1. (C) Consumption of monomeric wt αS (black), A53T αS (cyan), A30P αS (purple), A56P αS (yellow) and TP αS (red) monitored through 1D 1H NMR spectroscopy. Drop in signal intensity is due to the formation of higher molecular weight aggregates not detectable by solution-state NMR. Errors were estimated from three independent aggregation assays. (D) Fibril formation of wt αS (black), A30P (purple), A53T (blue), A56P αS (yellow) and TP αS (red, also shown in Inset a at a different scale) followed by ThT fluorescence emission intensity. Inset b shows ThT intensity values of all but TP variants, each of them normalized by the maximal value observed along their aggregation reaction. Download figure Download PowerPoint In agreement with the known β-breaking propensity of proline residues, the proline mutations markedly slowed down fibril formation. Although 0.1 mM samples of wt and A30P αS formed fibrils after about 20–30 h, as probed by thioflavin T (ThioT) fluorescence, A56P, A30PA56P and A30PA76P αS had an approximately five time longer lag phase (Figure 1B and Supplementary Figure S1). In addition, their fibril elongation rate was strongly reduced, suggesting a reduced cooperativity of the transition. TP αS did not show any fibrils even after two weeks of incubation (Figure 1B). A quantitative analysis of the NMR signal decay showed that after 50 h of incubation at 37°C and 0.1 mM protein concentration, the oligomeric intermediates constituted a 6% and 2% fraction of the protein mixture for A56P and TP αS, respectively. In the case of TP αS, the oligomeric fraction increased to 4% after 160 h (Figure 1C). A mixture of oligomeric and monomeric TP αS, which was obtained after five days of aggregation of TP αS at 37°C and 0.2 mM protein concentration, was able to seed fibril formation of monomeric wt αS (Figure 2A). In contrast, addition of the same concentration of purely monomeric TP αS did not accelerate aggregation of wt αS. In addition, the mixture of oligomeric and monomeric TP αS was recognized by the conformation-specific antibody A11 (Figure 2B), which detects a variety of toxic amyloid oligomers (Kayed et al, 2003). It should be noted that the TP oligomers were not resistant to SDS (data not shown). Figure 2.Aggregates formed by TP αS seed aggregation of wt αS. (A) Pre-fibrillar aggregates of TP αS seed fibril formation of wt αS. In an equimolar mixture of pre-aggregated TP αS with monomeric wt αS the lag time of aggregation is decreased (light blue) when compared with wt monomer alone (black). In contrast, in an equimolar mixture of monomeric TP αS and monomeric wt protein the lag time is increased (dark blue). Aggregation behaviour of monomeric TP αS alone is shown in red. Error bars represent mean±s.d. of three to four independent experiments. (B) Recognition of a mixture of oligomers and monomers of TP αS (O/M) but not monomeric TP αS (M) on nitrocellulose membrane by the A11 antibody. Anti-αS antibody shows comparable attachment to both monomer and oligomer. Download figure Download PowerPoint Increasing the concentration to 0.8 mM significantly accelerated the rate and amount of aggregation, and amyloid formation of all αS variants (Figure 1D). At 0.8-mM protein concentration, wt and A30P αS had a distinct lag phase of about 9–12 h, whereas ThT reactivity of A53T rose from the beginning (inset in Figure 1D). However, A56P started to form ThioT-positive fibrils after about 72 h and TP αS showed a clear but very slow rising ThioT signal only after about 5 days of incubation (Figure 1D). Surprisingly, the strongest ThioT signal after 5 days of incubation was observed for A30P αS, followed by wt and A53T αS. This is most probably caused by the very gel-like behaviour of the aggregated wt, A30P and in particular A53T αS samples that interfered with ThioT binding. The samples of A56P and TP αS were much more fluid, indicating that a smaller amount of fibrils was formed. In addition, A56P and TP αS had strongly reduced fibril elongation rates. Although for wt, A30P and A53T αS it took about 20 h to reach the saturating ThioT signal from end of the lag phase, this time was increased to about 60 h in case of A56P αS. With TP αS a saturating ThioT signal could not be reached within the experimental time, indicating that it has an extremely slow fibril elongation rate (Figure 1D). Electron microscopy of wt, A30P, A53T and A56P αS samples after 6 days of incubation (protein concentration of 0.8 mM) revealed a high number of fibrils of about 8 nm in diameter and various lengths but without clearly observable oligomeric species. In case of TP αS, the fibrils were significantly lower in number, longer and frequently associated with oligomers of various shapes and sizes (Figure 3A and Supplementary Figure S2). Atomic force microscopy of the TP αS sample showed a similar picture, with the presence of fibrils of about 8 nm in diameter and oligomers of 20–100 nm in diameter (Figure 3B). Figure 3.Enhanced formation of soluble oligomers by αS variants. (A) Electron micrograph of TP αS solution in 50 mM HEPES, 100 mM NaCl (pH 7.4), 0.01% NaN3, incubated for 6 days at 37°C while being stirred at 200 r.p.m. The protein concentration was 0.8 mM, and the sample was diluted eightfold by buffer before EM imaging. (B) AFM image of TP αS solution. Conditions identical to (A). (C) Dynamic light scattering of αS variants, incubated for 11 days at the aggregation condition and then centrifuged briefly followed by the measurement of the supernatant. Data presented are average of three measurements, each consisting of 20 acquisitions of 20 s. (D) UV absorbance of the supernatant of aggregated αS variants after 11 days of incubation at the aggregation condition. Download figure Download PowerPoint αS variants have an increased propensity to form oligomers The aggregation process of the αS variants was further investigated by dynamic light scattering and electron microscopy. Dynamic light scattering revealed the formation of soluble oligomers with a hydrodynamic radius of approximately 80–180 nm after six hours of incubation in the aggregation assay using protein concentrations of 0.1 mM (Supplementary Figure S3). In the same assay, a heterogeneous distribution of larger species was observed through electron microscopy for all αS variants after 12 h of incubation (Supplementary Figure S3). In addition, DLS was used to study the soluble oligomers of αS, which were formed after 11 days of incubation. At protein concentrations of 0.8 mM, fibrils were observed for all αS variants (see above and Figure 3A). The fibrillar material was separated from soluble oligomers by centrifugation at 14 000 g for 30 min and careful pipetting of the upper 50% of the supernatant. DLS measurements of the supernatant samples showed quite different scattering patterns for the different αS variants. The smallest scattering intensity was observed for wt αS (Figure 3C). A30P and A53T αS had very similar scattering intensities, which were slightly larger than those of wt αS, and for A56P αS the scattering intensity was further increased. The most marked increase, however, was seen for TP αS, for which the scattering intensity of the supernatant sample was an order of magnitude higher than in case of the wt protein (Figure 3C), and mostly caused by 140–170 nm oligomeric species. The UV absorbance spectrum of the supernatant showed a very similar trend for the αS variants (Figure 3D). The combined EM, AFM, DLS and UV data indicate that A56P and, in particular, TP αS have an impaired ability to form amyloid fibrils, but soluble oligomers accumulate in later stages of the aggregation. Aggregates formed by αS variants have impaired β-structure The late-stage aggregates of A56P and TP αS were characterized at single residue resolution through solid-state NMR (ssNMR) spectroscopy (Figure 4). For both A56P and TP αS, magnetization transfer from water to the protein proceeded at the same rate, suggesting that the relative water-accessible surface in these fibrils was similar to that of fibrils from wt protein (Figure 4A). Following a theoretical analysis, given in (Ader et al, 2009), and assuming a cylindrical (proto)fibril model, we estimated fibril diameters of about 60 Å for all three cases. However, cross peak signals in sequential (13C,13C) correlation experiments conducted on A56P αS were absent around the mutation site (e.g. Y39, S42, T54 and A56) but were identified for the other three β-strand segments previously detected for the wt, A30P and A53T protein (Heise et al, 2005, 2008; Zhou et al, 2006) (Figure 4B). In addition, we detected alterations in chemical shifts for several residues, including T75, Q79, I88 and E83, indicative of a perturbed β-strand structure. These findings point to a reduced β-sheet content in late-stage aggregates of A56P αS compared with wt, A30P and A53T αS. Concomitantly, we detected, in ssNMR experiments probing mobile A56P fibril segments, an enhanced contribution from residue types such as threonine, which are found in the residue stretch 22–93 (Figure 4D). For TP αS, a further significant reduction of cross-peak correlations was detected (Figure 4C) under experimental conditions comparable with A56P, consistent with structural alterations or increased dynamics/disorder in the last two β-strands (Figure 4E). Figure 4.High-resolution solid-state NMR of late stage aggregates formed by αS variants. (A) Water accessibility of aggregates as probed by solid-state NMR. A 3-ms Gaussian pulse and a T2 filter containing two delays of 1 ms were used for selective water excitation (Ader et al, 2009). The cross polarization contact time was set to 700 μs. (B) Superposition of 2D 13C/13C correlation spectra of U-[13C,15N] A56P αS (yellow) and of wt αS (black). Correlations absent in the A56P mutant are underlined. Assignments correspond to values obtained for the A form of wt αS as reported in (Heise et al, 2005). (C) Superposition of 2D 13C/13C correlation spectra of U-[13C,15N] A56P αS (yellow) and of U-[13C,15N] TP αS (red). In (B) and (C), homonuclear mixing was achieved using a proton driven spin diffusion time of 20 ms (A56P) and 50 ms (TP), respectively. (D) 2D 1H/13C 1H-T2-filtere