Redox Biology 22 (2019) 101135 Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research Paper Biasing the native α-synuclein conformational ensemble towards compact T states abolishes aggregation and neurotoxicity Anita Carijaa,b, Francisca Pinheiroa,b, Jordi Pujolsa,b, Inês C. Brásc, Diana Fernandes Lázaroc, Carlo Santambrogiod, Rita Grandorid, Tiago F. Outeiroc,e,f, Susanna Navarroa,b, ⁎ Salvador Venturaa,b, a Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain b Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medicine Göttingen, Waldweg 33, 37073 Göttingen, Germany d Department of Biotechnology and Biosciences, University of Milano-Bicocca, Italy e Max Planck Institute for Experimental Medicine, 37075 Göttingen, Germany f Institute of Neuroscience, The Medical School, Newcastle University, Framlington Place, Newcastle Upon Tyne NE2 4HH, UK A R T I C LE I N FO A B S T R A C T Keywords: The aggregation of α-synuclein (α-syn) into amyloid fibrils is a major pathological hallmark of Parkinson's α-synuclein disease (PD) and other synucleinopathies. The mechanisms underlying the structural transition of soluble and Disulfide bond innocuous α-syn to aggregated neurotoxic forms remains largely unknown. The disordered nature of α-syn has Amyloid hampered the use of structure-based protein engineering approaches to elucidate the molecular determinants of Protein aggregation this transition. The recent 3D structure of a pathogenic α-syn fibril provides a template for this kind of studies. Parkinson's disease The structure supports the NAC domain being a critical element in fibril formation, since it constitutes the core of the fibril, delineating a Greek-key motif. Here, we stapled the ends of this motif with a designed disulfide bond and evaluated its impact on the conformation, aggregation and toxicity of α-syn in different environments. The new covalent link biases the native structural ensemble of α-syn toward compact conformations, reducing the population of fully unfolded species. This conformational bias results in a strongly reduced fibril formation propensity both in the absence and in the presence of lipids and impedes the formation of neurotoxic oligomers. Our study does not support the Greek-key motif being already imprinted in early α-syn assemblies, discarding it as a druggable interface to prevent the initiation of fibrillation. In contrast, it suggests the stabilization of native, compact ensembles as a potential therapeutic strategy to avoid the formation of toxic species and to target the early stages of PD. 1. Introduction believed to be involved in the regulation of synaptic vesicle release [5]. In vitro, in its monomeric form, α-syn is an intrinsically disordered Parkinson's disease (PD) is the second most prevalent human neu- protein (IDP), whose primary structure is composed of the N-terminal rodegenerative disorder and it is pathologically characterized by the domain (residues 1–60), the non-amyloid-β component (NAC) (residues accumulation of intracytoplasmic aggregates in dopaminergic neurons. 61–95) and the C-terminal domain (residues 96–140). These inclusions are mainly composed of the protein α-synuclein (α- The central hydrophobic NAC domain was first observed in amyloid syn) and are known as Lewy bodies and Lewy neurites [1–4]. α-syn is plaques associated with Alzheimer's disease [6] and has been shown to present in high concentration at presynaptic terminals and it is found be a crucial region for the aggregation of α-syn [7,8]. Indeed, deletion both in soluble and membrane-associated brain fractions. The biolo- of certain residues within the NAC region abolishes α-syn aggregation gical role of this protein has not yet been completely elucidated, but it is [9,10] and peptides derived from the NAC domain can spontaneously Abbreviations: α-syn, α-synuclein; PD, Parkinson's disease; NAC, non-amyloid β-component; α-synCC, disulfide bridge-containing variant; CD, circular dichroism; Th-T, Thioflavin-T; CR, Congo Red; TEM, Transmission electron microscopy; PK, proteinase K; DOPC, dioleoylphosphatidylcholine; DMPS, dimyristoylpho- sphatidylserine ⁎ Correspondence to: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Parc de Recerca UAB, Mòdul B, E-08193 Bellaterra, Barcelona. E-mail address: salvador.ventura@uab.es (S. Ventura). https://doi.org/10.1016/j.redox.2019.101135 Received 20 October 2018; Received in revised form 18 December 2018; Accepted 4 February 2019 Available online 05 February 2019 2213-2317/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 1. Primary sequence and three-dimen- sional structure of wt α-syn and its double cysteine mutant α-synCC. (A) Primary se- quence alignment of wt α-syn and α-synCC. The amino acid sequences of wt α-syn (UniProt ID: P37840) and α-synCC are shown. The three distinct regions of α-syn are indicated. (B) Structural models of wt α-syn (left panel) and α-synCC (right panel) showing the NAC domain in purple. The models were generated in PyMol using the structure of pathogenic fibril of full- length human α-syn (PDB: 2N0A). Cys71 and Cys92 residues in α-synCC are shown in green. form β-sheet-rich amyloid fibrils [8,11] and are toxic to neuronal cells polymorphs, in vitro and in neuronal cells. We also monitored the im- [9]. pact of cross-linking the NAC domain on the lipid-mediated aggregation In addition to α-syn, there are other types of synucleins, β-syn and γ- of α-syn. Finally, we explored the influence of the introduced entropic syn, all sharing the highly conserved N-terminal domain [12]. β-syn has restriction on the conformation and neurotoxicity of α-syn oligomers. been reported to have a suppressing effect on α-syn aggregation in vitro and in vivo [13,14]. Interestingly enough, the most striking difference 2. Materials and methods between these two proteins is that β-syn lacks a NAC domain. Despite being evolutionarily close to α-syn, γ-syn has not been associated with 2.1. Design of α-syn disulfide bond the onset of PD [15]. Importantly, the γ-syn sequence stretch corre- sponding to the α-syn NAC domain is much less hydrophobic. The rational design of the disulfide bond in α-syn (α-synCC) was For long time, the structural heterogeneity and lack of persistent performed using Disulfide by Design 2.0 algorithm [21]. Two residues structural elements for α-syn has hampered the use of classical struc- within NAC region of α-syn, Val71 and Thr92, were mutated to cy- ture-based protein engineering approaches to elucidate the molecular steines to form a disulfide bridge. The stability of the mutated variant determinants of its aggregation and toxicity. The recent high-resolution α-synCC was assessed using FoldX [22]. The atomic spatial coordinates structure of a pathogenic fibril formed by full-length human α-syn of wt α-syn in the solved crystal structure of a pathogenic fibril of full- provides an outstanding template for this kind of studies [16]. This 3D length human α-syn (PDB: 2N0A) were used as an input. structure supports the NAC domain being a critical element in α-syn fibril formation, since it constitutes the core of the fibril, which consists 2.2. Analysis of aggregation propensity, disorder, and hydrophobicity of α- of a β-serpentine arrangement delineating a Greek key β-sheet topology syn sequences (Fig. 1). Some studies suggest that the structural elements of mature fibrils The sequential analyses were performed using web-based algo- are already imprinted in the early assemblies [17,18], conformationally rithms for predicting aggregation tendencies, such as AGGRESCAN biasing the aggregation pathway and the structural details of the final [23], Zyggregator [24] and FoldAmyloid [25]. All predictions were aggregate. In contrast, other works support that the structured regions performed using default settings. Intrinsic disorder for both wt α-syn in oligomers and fibrils are different [19], implying a conformational and α-synCC was analyzed using IUPred2 [26]. The hydrophobicity conversion as the aggregation reaction proceeds. Here, we exploited the values according to the scale of Kyte and Doolittle [27] were calculated reported fibrillar structure of human α-syn to engineer an artificial considering a 15-residue sliding window. disulfide bond within the NAC region. This cross-linking is expected to staple the Greek-key motif, conformationally constraining monomeric 2.3. Expression and purification of wt α-syn and α-synCC α-syn and potentially favoring the formation of this structural element early in the aggregation reaction. We compared the conformational pET21a vector (Novagen) encoding for wt α-syn was transformed properties of soluble wild type (wt) α-syn and its engineered disulfide into E. coli BL21(DE3) cells and the same vector encoding for α-synCC bridge-containing variant (α-synCC), as well as their aggregation re- was transformed into E. coli Origami(DE3) cells. actions and the structural properties of the aggregated states. Since α- wt α-syn and α-synCC were expressed and purified as previously syn aggregation process and final fibrillar structure are extremely sen- described [28]. The identity and purity of the recombinant proteins sitive to the environment [20], the assays were performed in three were assessed by MALDI-TOF on an Autoflex Speed mass spectrometer different solution conditions, resulting in the formation of different (Bruker Daltonik) and by SDS PAGE. 2 A. Carija et al. Redox Biology 22 (2019) 101135 2.4. Mass spectrometry spectropolarimeter Jasco-715 (Jasco Inc.). Spectra were recorded from 260 to 200 nm with 1 nm bandwidth, and a scan speed of 100 nm/min. The molecular mass of wt α-syn and α-synCC (resuspended in Average of ten accumulations were recorded for each spectrum. For bis- 50 mM ammonium bicarbonate), with or without a previous step of ANS, the samples were incubated with 10 µM bis-ANS, excited at reduction with 25 mM dithiothreitol (DTT), and after derivatization 370 nm using a Jasco FP-8200 spectrofluorometer (Jasco Inc.), and with iodoacetamide (IAA) was determined with a MALDI-TOF emission measured between 400 and 600 nm with slit widths of 5 nm. UltrafleXtreme spectrometer (Bruker Daltonik) using 2′,6′-dihydrox- yacetophenone acid as a matrix. Samples and matrix were mixed to 1:1 2.9. Aggregation assay ratio and 1 µL of the mixture was applied on a ground steel plate. The analyses were calibrated using external calibrants (Bruker Daltonik). From the prepared stocks of wt α-syn and α-synCC in buffer A, B and C, four aliquots of 300 µL of protein in each buffer were prepared to a 2.5. Reverse-phase high performance liquid chromatography (RP-HPLC) final concentration of 60 μM. Samples were incubated for 15 days in a analysis Thermomixer Comfort (Eppendorf) with 0.02% sodium azide at 600 rpm and 37 °C. In specific cases, 150 mM dithiothreitol (DTT), final α-synCC was resuspended in 50 mM Tris pH 8.4 supplemented with concentration, was added to the reactions. 100 mM NaCl buffer to a final concentration of 0.5 mg/mL, with and without 100 mM DTT, and analyzed by RP-HPLC in a Waters 2690 2.10. Conformational properties of wt α-syn and α-synCC aggregated in HPLC system (Alliance). Protein resuspended in DTT was incubated for different buffers 2 h and both samples were mixed with TFA 2% (v/v) to freeze the re- action before the injection. A linear gradient from 30% to 50% of buffer Amyloid fibril formation was monitored for 15 days by measuring B was applied to a standard C4 column (Phenomenex) 250 × 4.6 5 mm the binding of Thioflavin-T (Th-T) to the aggregated samples, using a at a flow rate 0.75 mL/min. Composition of the chromatography buffers Jasco FP-8200 spectrofluorometer (Jasco Inc.) with an excitation wa- is the following: Buffer A - pure water + 0.1% TFA; Buffer B – acet- velength of 445 nm, and an emission range from 460 to 600 nm. The onitrile (Teknokroma) + 0.1% TFA. intensity of the spectra at 488 nm was recorded as an indication of the β-sheet structures in the aggregates. Additionally, the transition of α- 2.6. Electrospray ionization mass spectrometry (ESI-MS) analysis syn from initial soluble form to aggregated state after 5 or 14 days incubation was assessed by measuring light scattering and Congo Red Mass spectra have been acquired on a hybrid triple quadrupole-TOF (CR binding). For light scattering, a Jasco FP-8200 spectrofluorometer instrument, QSTAR Elite (Applied Biosystems), equipped with a nano- (Jasco Inc.) was used, with an excitation wavelength of 330 nm and an electrospray source. The samples (10 µL of 10 µM protein in 20 mM emission range from 320 to 340 nm. CR binding was determined using a ammonium acetate pH 7.0) were injected by borosilicate-coated ca- Cary-400 Varian spectrophotometer (Varian Inc.) by monitoring the pillaries with emitter tips of 1 µm internal diameter (Thermo Fisher absorbance spectra from 375 to 675 nm. A final protein concentration Scientific). The reduced form of α-synCC was obtained by incubating of 10 µM was used for the three techniques. For Th-T and CR binding the sample at room temperature (RT) with 20 mM DTT for 2 h before the samples were supplemented with 25 µM Th-T and 10 µM CR, re- the injection. Spectra were acquired with the following instrumental spectively. Buffers without protein were used as negative controls for parameters: ion spray 1.1 kV; declustering potential 60 V; curtain gas Th-T and light scattering measurements. For CR, buffer B was used as a 20 PSI. Final spectra have been averaged over 1 min acquisition time. baseline, independently of the buffer in which the aggregates were formed. All measurements were carried out in four replicates. 2.7. NMR spectroscopy 2.11. Transmission electron microscopy (TEM) Samples of wt α-syn and α-synCC were prepared at a final con- centration of 160 µM in 20 mM HEPES pH 7.4, and further diluted using For TEM imaging, wt α-syn and α-synCC incubated for 5 and 14 a 9:1H2O/D2O ratio. 2D 1H–1H spectra of both proteins were collected days in buffer A, B and C, were diluted in Milli-Q water to a final on a Bruker Avance 600 MHz (Bruker). The 2D-TOCSY pulse sequence concentration of 20 µM. Prepared samples were adsorbed onto carbon- used was solvent suppression dipsi2etgpsi19. DIPSI-2 TOCSY mixing coated copper grids, washed with water and negatively stained with 2% sequence applied was 80 ms at a spectral resolution of 6 × 96 Hz (F2 x (w/v) uranyl acetate. The morphology of the aggregates was observed F1) (8.3 kHz). Accumulation of 32 scans was recorded in a total ac- using a JEM-1400 transmission electron microscope (JEOL) at an ac- quisition time of 80 min. DOSY NMR experiments were performed with celerating voltage of 80 kV and recording images in a CCD GATAN a pulsed-field gradient stimulated echo (ledbpgppr2s) using bipolar ES1000W Erlangshen camera (Gatan Inc.). gradients. The spectral resolution was 0.46 Hz. The evolution of the pulsed-field gradient during the NMR diffusion experiments was es- 2.12. Limited proteolysis tablished in 16 steps, applied linearly between 2% and 98%. Each NMR diffusion experiment was obtained from the accumulation of 128 scans. Protein samples were resuspended in corresponding buffers at a final concentration of 50 µM and incubated with 2.5 μg/mL proteinase 2.8. Conformational properties of soluble wt α-syn and α-synCC in different K (PK) (Sigma Aldrich) at 37 °C. Proteolytic aliquots were taken at buffers different time points and the reactions were quenched by addition of the same amount of 4 times concentrated denaturing loading buffer, The stocks of wt α-syn and α-synCC were prepared by dissolving resulting in a 143 mM final concentration of 2-mercaptoethanol in the lyophilized protein in buffer A (50 mM sodium acetate pH 5.0), buffer B buffer. Samples were heated at 99 °C for 10 min, and 10 µL of each (10 mM sodium phosphate pH 7.0), and buffer C (10 mM sodium sample was loaded on SDS-PAGE gels and stained with Coomassie-Blue. phosphate pH 7.0 supplemented with 200 mM NaCl). After filtering the Soluble protein in buffer B was digested in the same manner and used as stocks through 0.2 µm filter units, the samples were diluted to a final a control. concentration of 15 µM in the corresponding buffers and the far-UV circular dichroism (CD) spectra and fluorescence emission spectra of 2.13. Secondary structure determination bis-ANS were obtained. Far-UV CD spectra were acquired in a thermostated ATR-FTIR spectroscopy analysis of amyloid fibrils was performed 3 A. Carija et al. Redox Biology 22 (2019) 101135 using a Bruker Tensor 27 FTIR Spectrometer (Bruker Optics Inc.) with a concentrations of α-synCC (also in buffer B). Aggregation kinetics were Golden Gate MKII ATR accessory. Incubated samples were centrifuged performed as described in 2.15. and the insoluble fractions were resuspended in Milli-Q water. Each spectrum consists of 16 independent scans, measured at a spectral re- 2.17. Preparation and analysis of oligomer-rich samples of wt α-syn and α- solution of 4 cm−1, within the 1800–1500 cm−1 range. Second deri- synCC vatives of the spectra were used to determine the frequencies at which the different spectral components were located. Fourier-deconvolution The oligomeric assemblies of wt α-syn and α-synCC were prepared and determination of band position of the original amide I band were according to a previously described method [30] and morphologically performed using PeakFit software (Systat Software). and structurally characterized by TEM, light scattering, bis-ANS and ATR-FTIR spectroscopy. For each of these techniques, oligomers were 2.14. Internalization of wt α-syn and α-synCC aggregates in neuronal cells prepared as described in 2.11, 2.10, 2.8 and 2.13, respectively, and the same measurement conditions were used. Human neuroglioma (H4) cells were maintained in Opti-MEM I- To evaluate the toxicity of the oligomeric assemblies, human SH- GlutaMAX medium (Life Technologies-Gibco,Thermo Fisher Scientific) SY5Y neuroblastoma cells were cultured in F-12 medium supplemented supplemented with 10% fetal bovine serum Gold (FBS) (PAA) and 1% with 10% FBS, seeded in 96-well plates at a density of 4000 cells/well penicillin/streptomycin (PAN) at 37 °C and 5% CO2 atmosphere. 10,000 and maintained at 37 °C. Cells were incubated for 72 h in the presence cells/well were seeded in a 48 well-plate (Costar), 8 h prior to the ad- and in the absence (control) of 25 µM and 50 µM soluble oligomeric dition of α-syn species. For each condition, α-syn fibrils prepared from assemblies of wt α-syn and α-synCC and 20 µL of PrestoBlue® Cell a 1 µM solution of monomeric α-syn were added to the cell media. Viability Reagent (Invitrogen) was added. After incubating for 45 min, Equivalent volumes of the vehicle buffers were added to cells as ne- at 37 °C, fluorescence was recorded at 615 nm, with an excitation wa- gative control. Cells were treated with wt α-syn or α-synCC fibrils. After velength of 531 nm, in a Victor fluorescent plate reader (Perkin Elmer). 24 h, cells were washed with sterile DPBS (PAN), transferred to a new Experiments were carried out in four replicates. 48 well-plate and analyzed by immunocytochemistry after 24 h. The cells were fixed with 4% paraformaldehyde for 30 min at RT, and 3. Results permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 20 min. Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and in- 3.1. Design of a disulfide bond within α-syn Greek-key motif cubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C. After washing the cells, they were incubated The Disulfide by Design 2.0 algorithm [21] was used on top of the with secondary antibody (1:2000, Alexa Fluor 555 donkey anti-rabbit fibrillar structure of full-length human α-syn [16] (Fig. 1A) to create a IgG, Molecular Probes) for 1 h at RT, and labelled with phalloidin variant containing two Cys residues within the central hydrophobic (Alexa Fluor™ 488 Phalloidin, 0.165 µM, Molecular Probes) also for 1 h NAC region (α-synCC). We searched for two residues close enough in at RT. Nuclei were counterstained with DAPI (1:5000) (Life the Greek-key motif to favor the formation of a novel intramolecular Technologies) in 1xPBS. Imaging was performed in a Leica DMI 6000B disulfide bond when mutated to Cys (Fig. 1B). The computational (Leica Microsystems), and the images were processed and analyzed analysis indicated Val71 and Thr92 as the unique candidates, due to using ImageJ (NIH) and Imaris 7.1.5 software (Bitplane). their proximity (distance between the β-carbons = 3.97 Å), face-to-face orientation and proper dihedral angles in the bound state (calculated 2.15. Aggregation in the presence of lipid vesicles χ3 torsion angle = −100.9). Dioleoylphosphatidylcholine (DOPC) vesicles and dimyristoylpho- 3.2. Cys mutations in the NAC domain do not alter the predicted sphatidylserine (DMPS) vesicles (50:30:20 DOPE/DOPS/DOPC) (Avanti aggregation propensity, hydrophobicity or disorder of α-syn Polar Lipids), a lipid composition suggested to mimic the composition of synaptic vesicles, were prepared by the lipid hydration and extrusion Before proceeding with the experimental characterization, we method [29]. Briefly, stock lipids in chloroform were thoroughly mixed wanted to ensure that the mutations will not affect significantly the in a round glass tube. Organic solvents were removed to yield a lipid sequential properties of α-syn, in terms of predicted aggregation pro- film using a dry nitrogen stream. The dried lipids were then hydrated in pensity, hydrophobicity, or degree of disorder. This would allow us to buffer B. Multilamellar liposomes (MLV) were formed by three cycles of ascribe the differences in conformation, aggregation and toxicity of α- vigorously mixing and sonication. MLV were extruded 20 times through synCC, if any, univocally to the presence of the new disulfide bond. 200 nm polycarbonate membranes (Avanti Polar Lipids) in an extruder The AGGRESCAN [23], Zyggregator [24] and FoldAmyloid [25] device to obtain small unilamellar vesicles (LUV). The size of the lipid algorithms were used to estimate the effect of Val71Cys and Thr92Cys vesicles was determined by dynamic light scattering (Malvern) in order mutations. These amino acid changes are not expected to promote to have a homogenous sample. significant differences in the intrinsic aggregation propensity of α-syn For testing the effect of LUVs on the aggregation kinetics of wt α-syn (Fig. 2A). and α-synCC, lyophilized protein was prepared in buffer B to a final A sequence analysis using the Kyte and Doolittle scale [27] showed concentration of 70 µM supplemented with 10 µM Th-T, and 70 µM that wt α-syn and α-synCC hydrophobicity profiles overlap, confirming DOPC or DMPS vesicles. The samples were transferred to a 96-well the NAC region as the most hydrophobic (Fig. 2B). plate, a 1/8′′ diameter teflon ball (Polysciences Europe GmbH) was To estimate if the mutations would impact wt α-syn sequence in- added to each well and the plates were incubated for 72 h at 37 °C on a trinsic disorder propensity, we used the IUPred2 algorithm [26], an 96-well Victor Microplate reader (Perkin Elmer). During this time, the update of IUPred [31] that allows to model the contribution to disorder samples were kept in quiescent conditions and agitated for 10 s each of Cys residues in their unbound state. The sequence of α-synCC was 15 min, just before Th-T fluorescence at 486 nm was measured. predicted to be as disordered as the one of wt α-syn (Fig. 2C). Experiments were carried out in four replicates. 3.3. Cross-linking the α-syn NAC region 2.16. Impact of α-synCC on the aggregation of wt α-syn We cloned, produced, and purified α-synCC. We analyzed soluble wt Lyophilized wt α-syn was prepared in buffer B to a final con- α-syn and α-synCC using MALDI-TOF MS. The absence of any detect- centration of 70 µM supplemented with 10 µM Th-T and different able alkylation upon treatment of α-synCC with vinylpyridine already 4 A. Carija et al. Redox Biology 22 (2019) 101135 To further confirm the presence of the disulfide bond, α-synCC was incubated in 25 mM DTT prior to alkylation with IAA (Supplementary Fig. 1B). The resulting peak shifted to 14,578.5 Da. This mass shift of 114.4 Da corresponds to the alkylation of the two Cys with two IAA molecules (57.05 Da). Addition of IAA to oxidized α-synCC or to wt α- syn did not promote any detectable shift in MW (Supplementary Fig. 1A and B). We could not detect the presence of any intermolecularly Cys- bonded species for α-synCC. 3.4. NAC cross-linking promotes α-syn compaction Monomeric α-syn has been shown to populate a wide range of conformations in solution, including compact, but transient species resulting from long-range interactions within the fluctuating structural ensemble. The establishment of a disulfide bond would reduce the configurational entropy of the protein ensemble (ΔS) [32,33]. The loss in ΔS is proportional to the length of the closed loop (N), which in the case of α-synCC corresponds to 20 residues, and can be approached with the following expression: 3 ΔS = −2.1−⎛ ⎞ RlnN ⎝2⎠ Thus, the disulfide bond in α-synCC is expected to promote a de- crease in ΔS of 11.0 cal/mol·K relative to wt α-syn, accounting for a total conformational entropy loss of 3.4 Kcal/mol at 25 °C, thus re- sulting in less flexible and more compact conformations. The predicted impact in protein compactness was first assessed using RP-HPLC. Both α-synCC and its reduced form were analyzed (Fig. 3A). Oxidized α- synCC eluted earlier than reduced α-synCC, indicating that, as ex- pected, hydrophobic residues have a lower capacity to interact with the column when the protein is in the disulfide-bonded state. To confirm the compaction of soluble α-synCC we used ESI-MS under non-denaturing conditions, which permits monitoring con- formation and oxidation distinctly and simultaneously in a physiolo- gically relevant environment (Fig. 3B, C and 3D). Mass determination informs about the oxidation state, whereas charge-state distributions allow detection of coexisting conformational species. Again, the mea- sured molecular masses were in excellent agreement with the theore- tical ones. MW of (14,459.8 ± 0.3) Da, (14,464.4 ± 0.3) Da and (14,466.1 ± 0.3) Da were obtained for wt α-syn, oxidized α-synCC and reduced α-synCC, respectively. The effect of the disulfide bridge in α- synCC can be analyzed on 4-component Gaussian fits of the spectra [34,35]. Cross-linking results in the accumulation of a compact inter- mediate state (green curve), which increases from (23 ± 3) % to (68 ± 8)% at the expenses of extended intermediate state (cyan curve) and fully extended conformer (magenta curve). The overall average charge state shifts from (16.0 ± 0.3) to (14.2 ± 0.3). Under reducing conditions (20 mM DTT), the conformational ensemble of the mutant is similar to that of wt α-syn, with a decrease of the compact intermediate Fig. 2. Sequence analysis of wt α-syn and α-synCC. (A) Aggregation pro- to (19 ± 3)% and of the average charge state to (15.7 ± 0.3). The pensity analysis using different sequential predictors: AGGRESCAN, content in compact conformers in α-synCC is 2.6 fold higher than in wt Zyggregator and FoldAmyloid. (B) Hydrophobicity plots. Residues exhibiting values above 0 are considered to be hydrophobic. (C) Disorder prediction with α-syn. On the contrary, the proportion of low compact and fully ex- IUPred2. Residues exhibiting values above 0.5 are considered as disordered. In tended conformations is 3.0 fold higher in wt α-syn compared to α- all plots, wt α-syn (black) and α-synCC (red). synCC. These data indicate unequivocally that the observed compaction is caused by the covalent bond. We also analyzed the soluble states of wt α-syn and α-synCC using suggested that the designed Cys71-Cys92 disulfide bond was sponta- homonuclear two-dimensional NMR (2D NMR) spectroscopy. neously formed in cells. The MW of wt α-syn and α-synCC were Unfortunately, we could not use heteronuclear MulD NMR spectroscopy 14,460.6 Da and 14,464.9 Da, respectively. For wt α-syn, the experi- with stable isotope labelling, due to the inability of E. coli Origami B mental (14,460.6 Da) and theoretical (14,460.0 Da) average masses (DE3) cells, the expression strain for α-synCC, to grow in minimal coincide (Supplementary Fig. 1A). This is not the case for α-synCC, for medium. The 2D 1H-1H spectra of both proteins exhibit the narrow which the difference between the experimental (14,464.9 Da) and chemical shift dispersion characteristic of natively unfolded polypep- theoretical (14466.1 Da) average masses (Supplementary Fig. 1B) can tides (Fig. 3E). However, there is small signal overlap between both be ascribed to the two protons in the sulfhydryl groups (-SH) of Cys spectra, confirming again that the soluble states of wt α-syn and α- residues, present in the reduced form but absent in the oxidized state. synCC are conformationally distinct. 5 A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 3. α-synCC disulfide formation induces compactness. (A) RP-HPLC elution profile of α-synCC before (black) and after reduction with DTT (red). (B-D) ESI- MS analysis showing the conformational ensembles of wt α-syn and α-synCC. ESI-MS spectra and Gaussian fits of the charge-state distributions obtained using 4 conformers (orange - compact conformer; green - compact intermediate state; cyan - extended intermediate state; magenta - fully extended conformer) for wt α-syn (B), oxidized α-synCC (C) and reduced α-synCC (D) are shown. The most intense peak of each ESI-MS spectra is labelled by the corresponding charge state. Inserts report the mass-deconvolution spectrum. Each Gaussian component is labelled by its relative amount in percentage. (E) 2D-TOCSY 1H–1H NMR-spectrum of wt α-syn (black) and α-synCC (red) dissolved in 20 mM HEPES, pH 7.4 at 25 °C. 3.5. Conformational properties of wt α-syn and α-synCC under different fluorescent probe bis-ANS. solution conditions Deconvolution of the CD spectra of freshly dissolved proteins at 15 µM indicates that wt α-syn and α-synCC do not exhibit significant The control of solution conditions can be employed to generate α- differences in secondary structure content in any of the solutions syn amyloid assemblies with different structural characteristics that (Fig. 4A). In all cases, they display CD spectra consistent with an es- behave as strains, displaying different seeding propensities and pro- sentially disordered/random coil conformation. ducing distinct histopathological and behavioural phenotypes [20,36]. In buffer A, both wt α-syn and α-synCC promote high bis-ANS Therefore, we decided to evaluate the aggregative properties of wt α- fluorescence (Fig. 4B). α-synCC promoted higher bis-ANS fluorescence syn and α-synCC under three different solution conditions. Both the emission than wt α-syn in buffers B and C. For both proteins, the solution pH and salt content have been shown to influence the ag- fluorescence emission of the dye was much lower at neutral pH than at gregation of α-syn. Thus, we chose to use the following conditions in pH 5.0, thus indicating that they exhibit higher exposure of hydro- our assays: 50 mM sodium acetate pH 5.0 (buffer A); 10 mM sodium phobic patches at acidic pH. phosphate pH 7.0 (buffer B) and 10 mM sodium phosphate supple- mented with 200 mM NaCl pH 7.0 (buffer C). The use of these buffers 3.6. Kinetics of wt α-syn and α-synCC aggregation would allow us to monitor the influence of the pH (buffers A and B) and the effect of ionic strength at neutral pH (buffers B and C), simulta- For aggregation studies, wt α-syn and α-synCC at 60 µM final con- neously. centration were incubated in the three different buffers at 37 °C with Before initiating the study of the aggregation reactions, we assessed agitation (600 rpm). The aggregation process was monitored during the properties of the initially soluble states using far-UV CD and the fifteen days by measuring the binding of the protein to the amyloid- 6 A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 4. Characterization of soluble wt α-syn and α-synCC. (A) Far-UV CD spectra of wt α-syn (dashed line) and its double cysteine mutant (full line). Soluble proteins at 15 µM were measured in buffer A (black), B (blue) and C (green). (B) Bis-ANS fluorescence spectra of 10 µM bis-ANS bound to 15 µM soluble wt α-syn and α-synCC in buffer A, B and C. specific dye Th-T. fluorescence was always higher in buffer B than in buffer C. Striking differences between the two proteins in each given condi- tion and for each individual protein under different conditions were observed (Fig. 5). Incubation in buffer A results in the appearance of wt 3.7. Conformational and morphological characterization of wt α-syn and α- α-syn Th-T positive species already in day 1, whereas in buffers B and C synCC aggregation reactions their formation was significantly delayed. When incubated in buffer A, α-synCC did not bind to Th-T along the reaction. In contrast, incubation To gain insight into the species populating the different aggregation of α-synCC in buffers B and C results in the formation of Th-T positive reactions, samples of wt α-syn and α-synCC incubated for 5 days at assemblies, although the observed Th-T fluorescence emission was 37 °C were characterized by light scattering, Th-T fluorescence and CR lower than that of wt α-syn at any time point. For α-synCC, Th-T binding (Fig. 6A, B and C). In buffer A, the light scattering of the wt α- syn solution was significantly higher than that of the mutant; this came together with a higher binding to both Th-T and CR. Indeed, α-synCC exhibited negligible binding to Th-T and CR. In buffer B, wt α-syn and α-synCC solutions exhibited similar light scattering. Both bind Th-T and CR; however, the binding to Th-T was around 2 times higher for wt α-syn than for α-synCC. In buffer C, the light scattering and Th-T binding exhibited by wt α-syn were sig- nificantly higher than for α-synCC. To ensure that the reduced Th-T binding of α-synCC in buffers B and C owes to the presence of the disulfide bond, we incubated the protein for 5 days at 37 °C in the ab- sence and presence of the reducing agent DTT (150 mM). As expected, in both buffers, Th-T binding is significantly higher under reduced conditions (Supplementary Fig. 2). Consistent with Th-T measurements, analysis of the solutions con- tent by TEM (Fig. 6D) demonstrated that wt α-syn forms amyloid fibrils in all three buffers, while α-synCC is unable to aggregate into amyloid structures at acidic pH. Importantly, differences in the morphology of the fibrils were observed depending on the aggregation conditions. wt Fig. 5. Aggregation kinetics of wt α-syn and α-synCC in three different α-syn fibrils in buffer A appear as very large bundles. In buffer B, fibrils buffers. The aggregation kinetics were monitored by following the change in formed by wt α-syn and α-synCC were long and straight, although those relative Th-T fluorescence signal during 15 day-incubation of 60 µM protein in formed by α-synCC seemed to be more structured. In the presence of buffers A (black), B (blue) and C (green). salt, the fibrils formed by wt α-syn were shorter than those formed in its 7 A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 6. Characterization of wt α-syn and α-synCC aggregates formed after 5 day-incubation in buffer A, B and C. (A) Static light scattering of 10 µM wt α-syn (dashed line) and α-synCC aggregates (full line) in different buffers: A (black), B (blue) and C (green). (B) Fluorescence emission spectra of Th-T upon incubation with 10 µM wt α-syn and α-synCC fibrils formed in different buffers. (C) CR normalized absorbance spectra in the presence of 10 µM α-syn aggregates. Free CR absorbance spectrum is represented in grey. (D) TEM micrographs of negatively stained aggregates formed by wt α-syn (upper panel) and α-synCC (lower panel) in the three different conditions. Scale bars represent 200 nm. Images were taken at a 10,000× magnification. (E) PK degradation patterns of soluble wt α-syn and α-synCC (panels without frames), together with the digestion pattern of the aggregated forms (framed panels), monitored over time on Coomassie-stained SDS-PAGE. The time of digestion (in min) is indicated on the top of each panel. (F) The secondary structure of wt α-syn and α-synCC aggregates formed after 5 days. ATR-FTIR absorbance spectra in the amide I region was acquired and the fitted individual bands after Gaussian deconvolution are indicated. 8 A. Carija et al. Redox Biology 22 (2019) 101135 absence. α-synCC incubated in the same condition forms short, discrete, Supplementary Fig. 4). For proteins prepared in buffer B, the inclusions and straight structures. were predominantly of medium size for wt α-syn (Fig. 7B), while in the To explore whether the observed differences in fibril morphology case of α-synCC the proportion of medium and large deposits was si- were related to differences in the interactions supporting their archi- milar (Fig. 7E and Supplementary Fig. 4). Cells treated with the two α- tecture, we used limited proteolysis (Fig. 6E) and ATR-FTIR (Fig. 6F). syn variants generated in buffer C displayed mostly small inclusions The digestion patterns of the assemblies were different (Fig. 6E), (Fig. 7C and F and Supplementary Fig. 4). As expected, no α-syn signal which together with the TEM images suggests that these assemblies was observed in cells treated with vehicle (supplementary Fig. 5). correspond to different α-syn strains. The large wt α-syn fibrillar bun- dles formed in buffer A were essentially resistant to PK digestion, 3.9. Impact of α-synCC on the aggregation of wt α-syn whereas α-synCC was very susceptible to the PK action, likely because a large fraction of the protein corresponds to soluble species. On the To test the effect of α-synCC on wt α-syn fibrillization, we set up a contrary, the fibrils formed by α-synCC in buffer B seemed to be more series of wt α-syn in-plate aggregation reactions in the absence and resistant than those of wt α-syn, exhibiting a different digestion pattern. presence of increasing concentration of α-synCC in buffer B (Fig. 8A The fibrils formed by both proteins in buffer C demonstrated high and B). This plate assay exploits Teflon balls for agitation and allows to sensitivity to proteolysis. monitor the changes in Th-T fluorescence emission of all the reactions To assess the structure of these assemblies, we analyzed the amide I continuously and simultaneously during 40 h. α-synCC interfered with region of the ATR-FTIR spectrum (1700–1600 cm−1). This region cor- the aggregation kinetics of wt α-syn even at substoichiometric ratios. responds to the absorption of the carbonyl in the peptide bond and it This effect was evident at the early stages of aggregation (Fig. 8B). The constitutes a marker of the protein secondary structure. After decon- presence of α-synCC increases the lag time of wt α-syn aggregation volution of ATR-FTIR spectra, the contribution of the individual sec- reaction in a concentration-dependent manner (Fig. 8C). Indeed, even if ondary structure elements to the main absorbance signal was assigned the reactions in the presence of α-synCC progress towards the formation (Fig. 6F and Supplementary Table 1). The secondary structure content of Th-T positive assemblies, inspection of these structures at the end of of wt α-syn fibrils in the three buffers was similar, exhibiting a band at the reaction by TEM indicates that the aggregates become increasingly 1628–1631 cm-1 attributed to the presence of amyloid-like inter-mole- disordered with increasing concentrations of α-synCC, rendering very cular β-sheet structure, a band at 1645–1647 cm-1 corresponding to short protofibrillar aggregates at equimolar wt α-syn: α-synCC con- disordered conformations, and two bands at 1661–1663 cm-1 and centrations (Fig. 8D). 1680–1682 cm-1, indicative of β-turns. For α-synCC assemblies, the spectra in buffer A is dominated by a band at 1654 cm−1 attributed to 3.10. Aggregation of wt α-syn and α-synCC in the presence of lipids random coil/helical conformations. In buffer B, the fibrils exhibit a strong signal at 1629 cm-1, indicating the presence of intermolecular β- α-syn exists in a dynamic equilibrium between a soluble state and a sheet. This characteristic band is also observed in buffer C but in a membrane-bound state and the interaction between α-syn and lipid lower proportion. surfaces is believed to be a key feature for mediating its cellular func- To determine whether the conformational properties of α-syn as- tion and its toxicity. The NAC region has been implicated as a sensor of semblies evolve with time, samples of wt α-syn and α-synCC were the lipid properties that determines the affinity of α-syn membrane characterized in the same manner after 14 days of incubation under the binding [37]. same conditions (Supplementary Fig. 3). Overall, in buffer A, the We explored if the striking differences in aggregation properties amyloid-like signature is always higher for wt α-syn than for α-synCC. described above were also observable in the presence of lipids. To this Even after 14 days, α-synCC exhibits low light scattering, does not bind aim, the aggregation kinetics of wt α-syn and α-synCC at 70 μM in Th-T or CR. In buffer B, wt α-syn and α-synCC display similar light buffer B were monitored in the above described in-plate assay. scattering, Th-T and CR binding. In the presence of salt, wt α-syn fibrils Unilamellar vesicles of varying net charge and lipid compositions were still maintains higher amyloid-like signatures than α-synCC, in- employed as model membranes; in particular, neutral liposomes dependently of the used spectral probe. (DOPC) and neural liposomes (DMPS) were used in buffer B, with a TEM analysis (Supplementary Fig. 3D) demonstrates amyloid fibrils protein: lipid concentration ratio of 1:1. of wt α-syn formed in all three buffers, and fibrils formed by α-synCC in As in the previous in-tube assays, in buffer B and in the absence of buffer B and C. In buffers A and B, wt α-syn and α-synCC aggregates lipids, wt α-syn aggregates into Th-T positive species significantly faster retain essentially the same morphology as the one observed at 5 days, and to a larger extent than α-synCC (Fig. 9A). The final point of the whereas in buffer C the aggregates evolve towards longer and more aggregation kinetics was analyzed by TEM (Fig. 9B). wt α-syn forms ordered fibrils. long unbranched fibrils, while the fibrils formed by α-synCC are thinner With the exception of α-synCC incubated in buffer A, in the rest of and shorter. cases, the ATR-FTIR spectra is now dominated by a peak at For wt α-syn, the presence of both types of liposomes resulted in a 1625–1631 cm−1, attributed to the presence of amyloid-like inter- retardation of the exponential phase, but a significant increase in the molecular β-sheet structure, which contributes > 40% of the absor- final Th-T fluorescence (Fig. 9A). In the case of α-synCC, the presence of bance signal (Supplementary Fig. 3 and Supplementary Table 2.). unilamellar vesicles promoted a remarkable inhibition of the aggrega- tion reaction, with very low levels of Th-T fluorescence after 40 h 3.8. Internalization of wt α-syn and α-synCC aggregates in human cells (Fig. 9A). In the presence of DOPC the wt α-syn fibrils are well-struc- tured, whereas the α-synCC aggregates appear as long, thicker fila- To assess the internalization of wt α-syn and α-synCC aggregates ments (Fig. 9B). In the presence of neural liposomes, both wt α-syn and and the putative correlation between the in vitro and intracellular its disulfide bonded variant form similar straight thin fibrils (Fig. 9B). features of these aggregates, H4 cells were treated with 5 days-old ag- gregates for 24 h and then cultivated for an additional 16 h after re- 3.11. Conformational and toxic properties of wt α-syn and α-synCC moving the non-internalized protein. oligomers We observed that the cells incubated with wt α-syn aggregates prepared in buffer A displayed large (> 20 µm2), medium size It is suggested that soluble oligomers rather than insoluble amyloid (10–20 µm2) and small (< 10 µm2) intracellular inclusions in similar fibrils are the main elicitors of neurotoxicity in PD and other synu- proportions (Fig. 7A and Supplementary Fig. 4), whereas in cells in- cleinopathies [38,39]. The fact that the clinical symptoms of PD are cubated with α-synCC the inclusions were mainly small (Fig. 7D and manifested before the appearance of fibrillar deposits [40], and that, in 9 A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 7. Neuroglioma cells incubated with wt α-syn or α-synCC aggregates generated in the different buffers. Cells incubated with wt α-syn produced in buffer A (A), buffer B (B) and buffer C (C). Cells incubated with α-synCC in buffer A (D), buffer B (E) and buffer C (F). Scale bar: 20 µm. 10 A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 8. wt α-syn aggregation in the presence of α-synCC. (A) Th-T time course of fibrillization of wt α-syn in buffer B, in the absence (black line) and in the presence of increasing concentrations of soluble α-synCC (lines coloured in a gradient of blue). (B) The first 800 min of the reaction showing the aggregation lag phase. (C) Bar diagram representation of the lag time of the aggregation kinetics in the presence of increasing α-synCC concentration. Error bars indicate ± SE (n = 3). (D) TEM micrographs of aggregates generated at the final point of the monitored aggregation kinetics. Scale bars correspond to 200 nm. Images were taken at a 10,000x magnification. vitro, α-syn oligomers are more toxic than the fibrils [41], are both in observed. α-syn oligomers do not exhibit significant differences in light support of this hypothesis. scattering (Fig. 10C). The differences in bis-ANS binding are not sig- We generated wt α-syn and α-synCC oligomers, after 21 h of in- nificant either, even though it has been reported for different and un- cubation in 20 mM HEPES pH 7.4 and tested their toxicity for cultured related proteins that the binding to ANS-like dyes correlates with the neuroblastoma cells from the SH-SY5Y cell line. In agreement with the toxicity of aggregated species [42] (Fig. 10D). The ATR-FTIR spectra of previous reports, the oligomers formed by wt α-syn in these conditions wt α-syn and α-synCC oligomers in the amide I region shows a low were toxic [30]. However, the oligomers formed by α-synCC did not content of intermolecular β-sheet conformations (Fig. 10E and have any effect on the viability of neuroblastoma cells (Fig. 10A). Supplementary Table 3), with a high content of disordered conforma- We analyzed the morphological and conformational properties of tions and β-turns. The secondary structure content of both oligomers both assemblies to see whether they can explain their differential was similar, irrespective of the presence of the intramolecular disulfide toxicity. As visualized by TEM (Fig. 10B), oligomers appeared as bond in α-synCC. spheroidal polydisperse aggregates, with diameters ranging from 6 to 10 nm. In the case of α-synCC, larger oligomeric forms could also be 11 A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 9. α-syn aggregation in the presence of lipids. (A) Change in Th-T fluorescence intensity when 70 μM wt α-syn (dots) and α-synCC (triangles) were incubated in the absence (black; upper panel) and in the presence of 70 μM DOPC (grey; middle panel) or DMPS (dark blue; lower panel) in buffer B and 37 °C. (B) TEM micrographs of fibrils generated at the final point of the aggregation kinetics. Scale bars correspond to 200 or 500 nm. Images were taken at a 10,000× magnifi- cation, except wt α-syn (DOPC) and α-synCC (DOPC) images that were taken at 8000× and 6000×, respectively. 4. Discussion sequentially distant regions of the polypeptide chain, disulfide bonds favor compact conformations in the unfolded protein ensemble, de- The aggregation of α-syn into amyloid fibrils plays a major patho- creasing its entropy and making it less favorable compared with the physiological role in the development of PD and other synucleino- folded state. We rationalized that, in an analogous manner, stapling of pathies [3,43]. The heterogeneity of these neurological disorders seems the α-syn Greek-key edges by a designed disulfide bond would promote to lie on the distinct conformational characteristics of α-syn fibril the compaction of the NAC region, reduce its flexibility and the entropy polymorphs or “strains” [44,45]. Therefore, there is a strong interest in of the protein in the α-syn disordered soluble ensemble, favoring the elucidating the molecular basis of α-syn fibrillation and its structural population of the folded motif in case it is populated early in the as- polymorphism. The recent solid-state NMR atomic structure of a pa- sembly reaction. thogenic fibril of full-length human α-syn [16] provides a unique op- We successfully introduced a disulfide bridge between residues portunity to address these issues using structure-based protein en- 71–92 within the NAC region. This bond would connect the first and the gineering approaches. An outstanding feature of this α-syn fibrillar last β-strands of the Greek-key motif in the fibrillar structure. α-synCC structure is the presence of steric zippers stabilizing an orthogonal is purified exclusively in its intramolecularly oxidized form, which in- Greek-key topology that connects distant regions in the NAC domain of dicates that the two ends of the NAC domain come close in the space, at α-syn, a hydrophobic segment that is both necessary and sufficient for least transiently, in the bacterial cytosol. The bacterial cytoplasm con- fibril formation [10]. stitutes a reducing environment; therefore, in α-synCC the Cys residues The rational design of new disulfide bonds is a straightforward way should be significantly protected from the solvent in the disulfide to increase the stability of globular proteins [46,47]. By cross-linking bonded state. Computational analysis indicate that the identity of the 12 A. Carija et al. Redox Biology 22 (2019) 101135 Fig. 10. Evaluation of wt α-syn and α-synCC oligomers conformation and toxicity. (A) Cell viability of SH-SY5Y neuroblastoma cells after 72 h-incubation in the presence of different concentrations of soluble (S) and oligomeric (O) forms of wt α-syn and α-synCC. Error bars indicate ± SE (n = 3). (B) Electron micrographs of oligomeric assemblies of wt α-syn (dashed line) and α-synCC (full line) generated after 21 h of incubation in 20 mM HEPES, pH 7.4. The scale bars correspond to 200 nm. Images were taken at a 20,000x magnification. (C) Static light scattering of 10 µM wt α-syn (dashed line) and α-synCC (full line) oligomeric assemblies. (D) Bis-ANS fluorescence spectra of 10 µM bis-ANS bound to 15 µM wt α-syn and α-synCC oligomers in 20 mM HEPES, pH 7.4. (E) Secondary structure of wt α-syn (left panel) and α-synCC oligomers (right panel). ATR-FTIR absorbance spectra in the amide I region was acquired and the fitted individual bands after Gaussian de- convolution are indicated. introduced mutations does not change the sequential intrinsic proper- and remains mostly in a disordered conformation forming mostly small ties of α-syn. Thus, the observed differences in conformation, ag- aggregates, both in vitro and within cells. This indicates that a flexible gregation and cytotoxicity can be univocally ascribed to the effect of the and unconstrained NAC region is a pre-requirement for amyloid for- covalent link in α-synCC. mation and, thus, that neutralization of the α-syn charge by itself is not Soluble wt α-syn and α-synCC remain essentially disordered, but sufficient to promote aggregation, as previously assumed. RP-HPLC, ESI-MS and NMR data converge to indicate that they are In agreement with our MS data, paramagnetic relaxation enhance- conformationally distinct. As intended, the introduction of the disulfide ment and NMR dipolar couplings have shown that at neutral pH, native bond biased the conformational α-syn ensemble towards the population α-syn populates an ensemble of conformations comprising a continuum of more compact conformers. This results in dramatic differences in the of conformers ranging from highly unfolded to fairly compact [49]. The aggregation properties of both proteins. compact forms are stabilized by long-range interactions and it has been We could confirm that the solution conditions have a critical impact proposed that they would inhibit oligomerization and aggregation and in the aggregation rates, morphology and conformation of wt α-syn that only the fully unfolded species would allow the establishment of fibrils, resulting in different polymorphs. Both the pH and ionic strength the intermolecular contacts leading to protein aggregation, by ren- impact the aggregation reaction and the properties of the final product. dering the hydrophobic patches of the NAC region accessible to solvent. This phenomenon is more dramatic in the case of α-synCC. Importantly, The low aggregation propensity of α-synCC can be explained in this the two proteins exhibit very different aggregation properties even context, since upon the introduction of the disulfide bond the con- when they are under the same conditions. This indicates that, irre- formational ensemble becomes dominated by compact, potentially in- spective of the environment, the aggregation reaction of α-syn is hibitory, states. strongly influenced by the conformational bias of the initially soluble The presence of α-synCC has a significant, concentration-dependent protein ensemble. impact on the aggregation reaction of wt α-syn. Not only it delays the Under each tested condition, α-synCC exhibits reduced aggregation onset of the exponential phase, but it determines the structure of the propensity relative to wt α-syn, but the more striking differences are final aggregate. The inhibitory effect at substoichiometric ratios in- found at pH 5.0. For wt α-syn, the aggregation is much faster at pH 5.0 dicates that α-synCC interferes with the nucleation and/or elongation than at pH 7.0. Differences in wt α-syn aggregation rates at acidic and of wt α-syn fibrils. The presence of α-synCC biases the wt α-syn ag- neutral pH have been attributed to the acidic nature of the protein, gregation reaction towards the formation of short protofibrillar as- which results in a high net charge at neutral pH that is mostly neu- semblies. This suggests that the monomeric α-synCC state interacts with tralized at pH 5.0 (pI 4.7), making the protein more aggregation prone wt α-syn oligomeric nuclei and/or fibril ends, blocking the further or- [48]. In our assay, at pH 5.0 wt α-syn forms large β-sheet-rich fibrillar dered assembly of wt α-syn monomers on top of these species to form bundles that bind to amyloid dyes with high affinity, are resistant to long fibrils. proteolysis and accumulate in cells as large, medium and small ag- The binding of α-syn to lipid membranes is important for its func- gregates. In contrast, α-synCC is aggregation-incompetent at this pH tional role in synaptic regulation, but it may also trigger its aggregation 13 A. Carija et al. Redox Biology 22 (2019) 101135 [50]. Upon binding to lipid membranes, α-syn undergoes a significant References conformational transition and some regions adopt an α-helical struc- ture. It has been shown that this transient helical segment span residues [1] L. 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