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C-terminal truncation of α-synuclein promotes amyloid fibril amplification at physiological pH† †Electronic supplementary information (ESI) available: Supplementary methods, supplementary results, supplementary tables, supplementary figures. See DOI: 10.1039/c8sc01109e

C-terminal truncations shift the pH range at which α-synuclein secondary nucleation occurs from acidic to neutral values.


Introduction
Parkinson's Disease (PD) is a neurodegenerative disorder characterised by the presence of intracellular inclusions, called Lewy Bodies (LBs) and Lewy neurites, mainly composed of the protein a-synuclein (AS). [1][2][3] Although the physiological function of AS is not fully understood, the protein is primarily expressed at synaptic terminals in the central nervous system, 4 and is proposed to play a role in synaptic function and plasticity. [5][6][7] AS is estimated to represent $1% of the total protein content in soluble cytosolic brain fractions 4 and has also been found in mitochondria and at mitochondria-associated ER membranes. 8,9 AS is largely disordered in solution but can adopt an ahelical conformation when bound to biological and synthetic membranes. [10][11][12][13] The N-terminal region of AS consists of residues 1-60 and, together with the central segment (residues 61-95), contains seven imperfect repeats of 11 residues, which are characteristic of amphipathic helices. 10,14 Residues 61-95 form the NAC (non-amyloid b component) region which is characterised by the presence of hydrophobic residues 4 and is thought to form the core of AS amyloid brils. 15,16 The Cterminal region (residues 96-140) contains 14 acidic residues, giving it an overall negative charge, and remains exible and disordered when AS is bound to model membranes [10][11][12][13][17][18][19] (Fig. 1).
Monomeric AS is remarkably stable in solution at physiological concentration (50 mM) 20 under quiescent conditions at neutral pH, and does not detectably convert into amyloid brils unless mechanical agitation and/or specic interfaces, e.g. air/ water, 21 detergent/water, 22 lipid/water [23][24][25] or polystyrene/ water, [26][27][28] are introduced. It is now well established that the rst step in AS aggregation is the surface-induced nucleation at such interfaces, followed by the growth of the resultant species via monomer addition. 24,25,28,29 Secondary processes can be observed if mechanical agitation is used 24 or if the pH of the solution is decreased to 5.5 or below. 28,29 C-terminal truncation of AS is one of the predominant modications of AS associated with PD. 30 C-terminal truncated (Ctt) variants represent $15% of all AS molecules found in LBs 30 and are characterised by cleavage at positions in the vicinity of residue 120 [30][31][32][33][34][35][36] (see Fig. 1 and ESI, Table S1 †). Moreover, N-and C-terminally truncated variants have also been found in the brains of healthy controls. 37 Ctt variants of AS are thought to be generated from AS wild-type (WT) by processes that are independent of its aggregation both in brain tissues and in cultured cells 31 as a result of incomplete degradation by the 20S proteasome, 33,38 or by cleavage by calpain I, 39 prolyl oligopeptidase 40 and/or cathepsin D. 41 The different forms of Ctt AS have been found to be more prone to aggregation in vitro 33,[42][43][44][45] and in vivo 46 than AS(WT), showing a shorter lag phase, a higher level of bril formation and different aggregate morphology; the length of the lag phase in kinetic experiments generally decreases as more of the Cterminal region is truncated (see Table S1 †).
The disordered state of AS has been characterised as consisting of a broad distribution of conformers with a number of long-range interactions between the negatively charged Cterminal domain and the hydrophobic central region of the protein, 47 and between the N-and C-terminal domains. 48,49 It has been suggested that screening the negative charges at the Cterminal region of AS, by using small molecules, 43 by lowering the pH, 43 or by truncating parts of the C-terminus, 42,44 can lead to an increased aggregation propensity of the protein. These ndings suggest a protective role of the charges at the Cterminus against AS aggregation and that disruption of the long-range interactions via C-terminal truncations can facilitate aggregation. 50 In this study, we have investigated the inuence of Cterminal truncations on the different events involved in the process of AS aggregation. 24,29 We have found that deletion of Cterminal residues signicantly affects the properties of AS brils, including their ability to contribute to secondary processes. In particular, we have observed that truncation of segments of the C-terminus can induce a shi of the pH range at which secondary processes dominate the kinetics of AS aggregation, from acidic values for AS(WT) (3.6-5.6) to mildly acidic values for AS(1-119) (4.2-7.0) and to neutral values for AS(1-103) (5.6-8.0). Finally, we have found that C-terminal truncation of AS results in a change in the mechanism of lipid-induced aggregation as a result of the altered contributions of secondary processes to the overall mechanism of amyloid formation. The results suggest a critical role of the Cterminal acidic residues of AS both in secondary processes and in the conversion of AS protobrils into mature brils. Moreover, the dramatic shi of the pH region where secondary processes contribute to the overall kinetics of amyloid formation caused by C-terminal truncations suggests an explanation for the increased propensity of the C-truncated variants to aggregate in vivo.

Protein preparation
AS(WT), AS(1-119) and AS(1-103) were expressed and puried using essentially the same protocol as that reported previously, 24,29 with very minor alterations (see ESI for more details †).

Vesicle preparation
Small unilamellar vesicles (SUV) were prepared from DMPS using sonication as described previously. 24 Fibril formation at pH 3-9 Solutions containing ca. 260 mM of protein were incubated in phosphate buffer (20 mM H 3 PO 4 , 0.01% NaN 3 ) at pH values ranging from 3 to 9, adjusted using NaOH (Breckland Scientic, Stafford, UK). The solutions were stirred at 45 C for 48 h, then sonicated for 1 min (at 30% "on time", 10% power) using a Bandelin Sonopuls probe sonicator (HD2070) (Berlin, Germany) to homogenise the solution. At this stage, aliquots were taken for the preparation of samples for AFM and TEM (see details below). The rest of the solution was ultracentrifuged for 1 h at 90 krpm and 4 C (Beckman Coulter Optima™ TLX, High Wycombe, UK) and the supernatant was used to determine the concentration of soluble protein (using an extinction coefficient of 5600 M À1 cm À1 for AS(WT) and 1400 M À1 cm À1 for AS(1-119) and AS(1-103) at UV 278 nm (determined using the Scripps protein calculator at http://www.protcalc.sourceforge.net)).
Preparation of brillar seeds at pH 6.5 Solutions of 300 mM monomeric protein (AS(WT), AS(1-119) and AS(1-103)) were incubated in phosphate buffer (20 mM NaH 2 PO 4 /Na 2 HPO 4 , 0.01% NaN 3 , pH 6.5) (unless stated otherwise) at 45 C with maximum stirring for 50 h with sonication at 24, 48 and 50 h for 10 s (at 30% "on time", 10% power) (Bandelin Sonopuls HD2070). We also explored different sonication times and quantied the concentrations of soluble protein at the end of the 50 h incubation period (see ESI for more details †).
The resulting brils were used in experiments to measure elongation rates and these aliquots were diluted in phosphate buffer at pH 6.5, sonicated for 1 min (at 30% "on time", 10% power) and added immediately to solutions of monomeric protein.

Atomic force microscopy (AFM) measurements
Samples containing brils were diluted to give a bril concentration of 2 mM (monomer equivalent) in 20 mM phosphate buffer at the same pH as that of the sample. Diluted samples (30-50 ml) were deposited on cleaved mica (G 250-1, Agar Scientic, Stansted, UK), which was xed onto glass microscope slides (Thermo Scientic, Paisley, UK) and le to dry. Once dry, the slides were washed twice using 100-200 ml of doubledistilled water (Elga, High Wycombe, UK) and allowed to dry before storage at room temperature (RT).

Transmission electron microscopy (TEM) measurements
Samples containing brils were diluted to give a bril concentration of 5-10 mM (monomer equivalents) in 20 mM phosphate buffer at the same pH as that of the sample. Samples (5 ml) were deposited on grids and le for 1 min before being blotted using lter paper. The grids were washed once with double-distilled water, then incubated with 2% w/v uranyl acetate (Agar Scien-tic, Stansted, UK) for 45 s and nally washed again, twice, with double-distilled water. The grids were allowed to dry at RT, and imaged at the Cambridge Advanced Imaging Centre using a Tecnai G2 80-200 kV. Images were saved as 8-bit TIF les and processed using ImageJ. 52 The details of the grids used are as follows: C169/050 carbon support lm; thin clear lms of carbon 400 mesh 3 mm copper grid (TAAB Laboratories Equipment Ltd, Aldermaston, UK); C400Cu carbon lm, 400 mesh copper (EM Resolutions Ltd, Sheffield, UK), C169/N050 carbon support lm; thin clean lms of carbon, 400 mesh, 3 mm nickel grid (TAAB Laboratories Equipment Ltd).

Time-resolved aggregation kinetics
The changes in the ThT uorescence intensity during the different aggregation experiments described in this study were measured using multi-well plate-readers (POLARstar Omega, FLUOstar Omega, FLUOstar OPTIMA, CLARIOstar) from BMG Labtech (Ortenberg, Germany) using the bottom reading mode. Two types of plates were used in this work: (i) Corning® 96-well half area black with clear at bottom made of polystyrene treated with NBS™ (#3881, Corning Ltd, Corning, USA), referred to as "PEG-ylated plate" in the text, for the elongation and lipid-induced aggregation experiments (Fig. 2, 6, S12 and S13 †), and (ii) Corning® 96-well black with clear at bottom made of polystyrene with high-binding surface (Corning #3601), referred to as "polystyrene plate" in the text, for the non-seeded aggregation experiments performed at different pH values (Fig. 5, S8, S9 and S10 †).
Samples were incubated in the presence of 50 mM ThT (#88306, Anaspec Inc., Fremont, USA), unless stated otherwise, as triplicates or duplicates in the desired buffer under quiescent conditions. The wavelengths used were 440 nm (excitation)/ 480 nm (emission). Note: the stock solutions of ThT were prepared by dissolving ThT powder in phosphate buffer at the desired pH at about 1 mM nal concentration (w/v) and passed through a 0.22 mm syringe lter (SLGP033RS, Millipore, Darmstadt, Germany). The concentrations of samples generated by dilution in buffer were determined by absorbance spectroscopy using extinction coefficient of 36 000 M À1 cm À1 for absorbance at 412 nm (Abs 412,ThT ) 53 correcting for buffer background, using a CLARIOstar multi-well plate-reader.

Determination of insoluble protein concentration at the end of kinetic experiments
The concentrations of insoluble protein at the end of the aggregation reactions were determined using absorbance measurements such that [insoluble protein] ¼ [total protein] À [soluble protein], where [total protein] is the concentration of AS used at the beginning of the reaction and [soluble protein] is the concentration of protein soluble in the reaction mixture at the plateau phase. The reaction mixtures were collected and ultracentrifuged (Beckman Coulter Optima® TLX) for 30 min to 1 h at 90 krpm at RT. The supernatant was either stored at À20 C or directly transferred to a UV-transparent plate (Corning #3679) for absorbance measurements (CLARIOstar). The concentrations of soluble protein were calculated from the absorbance at 278 nm using the extinction coefficients given above, aer correcting for the buffer background signal. In the case of samples containing ThT, the absorbance of the supernatant measured at 278 nm (Abs 278,sup ) is the sum of the absorbance from ThT (Abs 278,ThT ) and that from the protein (Abs 278,protein ): Abs 278,sup ¼ Abs 278,ThT + Abs 278,protein . Abs 278,ThT was determined using the Abs 412,ThT and the following equation: Abs 278,ThT ¼ 0.147 Â Abs 412,ThT (see standard curve in Fig. S1 †).

Determination of ThT quantum yield
The ThT quantum yield was determined from the ratio of the ThT uorescence intensity to the concentration of insoluble protein at the plateau phase.

Circular dichroism (CD) experiments
Solutions of 20 mM protein were incubated in the presence of DMPS vesicles in phosphate buffer at pH 6.5 and 30 C. CD spectra were measured using a ChiraScan (Applied Photophysics, Leatherhead, UK) collecting ve repeats using a step size of 0.2 nm and a bandwidth of 0.5 nm. The CD spectra acquired for solutions containing 20 mM protein + 8 mM DMPS were analyzed using DichroWeb/CD Pro to extract the content of a-helix, b-sheet and random coil structure (input units: millidegrees/theta (machine units), K2D analysis program without a reference set). [54][55][56] The signals measured at 222 nm were acquired using the same equipment, collecting 50 repeats and bandwidth 0.5 nm. The binding curves were tted to a Langmuir isotherm as described previously. 24 Differential scanning calorimetry (DSC) experiments SUV made from DMPS at a concentration of 1 mM were incubated in the absence or presence of 100 mM protein in Eppendorf tubes and degassed for several minutes at RT before use in a MicroCal VP-DSC (Malvern, UK). DSC thermograms were acquired from 10-60 C at a scan rate of 90 C h À1 for buffer and 45 C h À1 for the samples under investigation.

Microscopy experiments in glass microcapillaries
Fibrils (100 mM, monomer equivalents) were rst sonicated for 1 min at 30% "on time" and 10% power and then diluted to a nal concentration of 5 mM in 20 mM phosphate buffer, pH 6.5, 50 mM ThT, in the absence or presence of 50 mM monomeric protein. The reaction mixtures were transferred through capillary action into capillaries (square boro tubing # 8250-050 with 0.50 mm internal diameter and 0.10 mm wall (CM Scientic, Silsden, UK)). Wax (Hawksley, Lancing, UK) was used to seal off the capillary ends and the capillaries were xed to standard glass microscopy slides (Thermo Scientic) using twocomponent epoxy resin (Araldite, Basel, Switzerland). The slides were xed to a heating plate set to 37.5 C in order to perform the experiments at or near the desired temperature of 37 C. A Zeiss inverted microscope (Axio Observer A1, Zeiss, Cambridge, UK) using multidimensional acquisition was used to observe uorescence intensity over time using a lter cube 49001 ET-CFP (excitation 426-446 nm, dichroic 455 nm, emission 460-500 nm). Images were taken every 20 s.

Results and discussion
C-terminal truncation of AS affects the properties of amyloid brils and decreases their seeding efficiency Amyloid bril formation occurs through a nucleated polymerisation mechanism and consists of distinct individual microscopic steps. 57,58 The elongation of brils is by far the fastest process in most cases and therefore it is responsible for the bulk of the formation of bril mass. 58 It is also a straightforward process to study experimentally in isolation, as pre-formed brils can be added to soluble protein in a well-dened manner ("seeding") and the resulting bril elongation can be measured with a variety of experimental techniques, such as ThT uorescence or biosensing techniques. 59,60 When the concentration of added seeds is high enough, all microscopic processes other than bril elongation can be neglected. We have previously performed such strongly seeded experiments for AS(WT), which has allowed us to determine the average molecular rate constant of bril elongation, 29 as well as its degree of heterogeneity. 61 In order to compare bril elongation rates of Ctt AS species to that of AS(WT), we performed seeded experiments using AS(WT), AS(1-119) and AS(1-103) brils and monomers. Preformed amyloid brils were incubated in solutions of monomeric protein molecules of the same variant (seeding) or one of the other variants (cross-seeding) (see Experimental section for details), and changes in ThT uorescence were used to monitor bril formation over time (Fig. 2). We found, as previously observed, 29 that when AS(WT) monomers were incubated in the presence of AS(WT) seeds, the ThT uorescence intensity increased rapidly, with a monotonically decreasing rate due to The linear fits to the low concentration data (solid lines) were used to obtain a robust measure of the elongation rate constant (see ESI for more details †).
the depletion of the soluble protein as the aggregation progresses (Fig. 2a). The concentration of insoluble protein present at the plateau phase of the reaction was found to correspond directly to the initial concentration of monomeric protein (Fig. 2b). This observation conrmed the fact that the majority of the monomeric protein molecules had been incorporated into brils 29 and provided a reference for comparison of equivalent experiments with the truncated variants.
In experiments in which the Ctt AS monomers were added to AS(WT) seeds (Fig. 2a, le column), the concentration of insoluble protein at the end of the reaction was found to correspond to the initial concentration of monomeric protein only for [Ctt AS] # 50 mM (Fig. 2b). This result suggests that Ctt variants can elongate AS(WT) seeds but that the elongation reaction only reaches completion for lower concentrations of Ctt AS under these conditions. We evaluated the rate by which the three AS variants elongate AS(WT) seeds at the different initial monomer concentrations (Fig. 2c) by analysing the slopes of the aggregation curves at early time points. In this way, we obtained the product, k + P 0 , of the elongation rate constant, k + , and the number concentration of seeds, P 0 , for each variant. By estimating P 0 from an analysis of the length distribution of the seeds at t 0 (ref. 29) ( Fig. S2 and S3, see ESI for more details †), we found that the elongation rate constant decreases as the degree of C-terminal truncation increases, from 3616 M À1 s À1 (AS(WT)) to 2779 M À1 s À1 (AS(1-119)) and 1963 M À1 s À1 (AS(1-103)). This nding suggests that the WT seeds do not provide as good a template for the truncated sequences as for the WT protein.
Alternatively, C-terminal truncation of the monomers could perturb the interaction of monomers with bril ends, as the successful attachment trajectory of a monomer to a growing bril is likely to involve contacts outside the regions that form the b-sheet core. Thus, although the C-terminal region is not being incorporated into the b-sheet core of the brils, it plays a role in dening absolute bril elongation rates.
We observed different behaviour when Ctt AS brils were used to seed the aggregation reaction ( Fig. 2a middle and right  columns). When Ctt AS seeds were incubated in solution in the absence of monomer, we observed an increase of up to a factor of two in the ThT-uorescence intensity, suggesting either that the bril samples initially contained unreacted or dissociated monomeric protein that was able to add onto the bril ends more efficiently aer the sonication step immediately prior to the experiment (see Experimental section for details), or that the binding of ThT to the brils made from the Ctt variant is slow compared to that to the WT brils. Additionally, when monomeric protein was added to solutions containing the Ctt AS seeds, we found that the kinetics of bril elongation were slower for brils containing the truncated variants. This nding was conrmed by the observation that the concentration of insoluble species formed when samples of the monomeric proteins (AS(WT), AS(1-119) and AS(1-103)) were incubated in the presence of Ctt AS seeds was much smaller than that formed under the same conditions using AS(WT) seeds (Fig. 2b).
Images of the seed brils prepared from the different AS variants, obtained using TEM and AFM, showed the presence of higher order assemblies for the Ctt AS (Fig. S2 †), suggesting that the accessibility of monomers to the ends of these seeds might be reduced by bril association. Such phenomena prevent an accurate estimation of the elongation rate constant, as the number of accessible growing bril ends is not likely to remain constant during the initial parts of the seeded aggregation curves. Due to this phenomenon, we only analysed quantitatively the data obtained by using AS(WT) seeds. Taken together, these results suggest that Ctt AS monomers elongate AS(WT) seeds at similar rates to those of AS(WT) monomers, but that Ctt AS brils are less able than AS(WT) brils to seed the aggregation of AS because of their higher propensity to cluster together.
Finally, we observed that the ThT uorescence yield, dened as the ThT uorescence intensity at the plateau phase per mole of insoluble protein, decreased with increasing values of concentrations of insoluble protein when AS(WT) brils were used to seed the aggregation reaction (Fig. S4a †). Furthermore, the ThT uorescence yield of brils formed from elongation of AS(1-103) seeds was found to be smaller for all concentrations of insoluble protein than that of those formed from elongation of AS(1-119) seeds (Fig. S4b †). The fact that we observed different ThT uorescence yields for the same concentrations of insoluble protein indicates that the binding of ThT to the brils is altered by the truncation.

C-terminal truncation of AS affects the pH-dependence of higher order assembly of amyloid brils
In order to investigate if the observed change in seeding efficiency and ThT uorescence yield associated with C-terminal truncations could be attributable to an alteration in the overall morphology of the brils formed, we monitored the seeding of AS aggregation in glass micro-capillaries under quiescent conditions at 37 C. This experimental procedure allows the observation of the higher order assembly behaviour of the aggregate species formed during the reaction 29 ( Fig. 3 and S5 †). When AS(WT) seeds were incubated in the absence of monomeric protein, the ThT uorescence was found to be essentially uniform throughout the capillary (relative standard deviation of $10%) and to remain approximately constant over time (Fig. 3). When AS(WT) seeds were incubated in the presence of AS(WT) monomers, we observed an increase of a factor of 1.4 in the mean ThT uorescence within the rst 4 h (Fig. 3); the relatively modest increase in ThT uorescence in this experiment can be explained by the time delay between the addition of the seed brils to the monomeric protein and the start of the measurements. If the ThT uorescence is compared with that of the sample containing only seeds, the relative increase in intensity as a result of the addition of monomer is 2.6 (Fig. 3).
The solution containing only Ctt AS seeds displayed a slightly (AS(1-103), relative standard deviation 14%) or much greater (AS(1-119), relative standard deviation 45%) degree of heterogeneity in the distribution of uorescence intensity along the capillaries (Fig. 3 and S5 †) relative to that observed for AS(WT). Addition of monomeric protein resulted in a similar average increase in uorescence intensity over time (2.6 fold for AS(1-119) and 2 fold for AS(1-103)) (see Fig. S5 † for images with increased brightness/contrast) but led to a more heterogeneous mixture (relative standard deviation of 68% for AS(1-103) and 61% for AS(1-119)) ( Fig. 3 and S5 †). The capillary experiments therefore suggest that the brils formed from the Ctt variants of AS have a stronger tendency than those of AS(WT) to form higher order assemblies.
In order to examine this conclusion further, the overall morphology and distribution of the brils formed by AS(WT), AS(1-119) and AS(1-103) was studied at higher resolution using TEM and AFM techniques over a range of pH values ( Fig. 4 and S6 †). Overall, we observed the maximal tendency of bril clustering to shi towards higher pH values from AS(WT) to AS(1-119) and AS . This trend can be rationalised in terms of the increase in the value of the isoelectric point (4.7 for AS(WT), 6.1 for AS(1-119) and 9.5 for AS(1-103) monomeric proteins) with increasing truncation of the largely acidic C-terminal domain. The isoelectric point of the brillar state can be expected to be similar, albeit not identical, to that of the monomeric state of AS; differences in the isoelectric point can be expected as a result of the more compact structure of the monomer inside the bril compared to the disordered soluble state. Bringing the brils closer to their isoelectric point decreases their net charge and destabilises the colloidal suspension of individually dispersed brils, leading to more pronounced higher order assembly process. 29 It is, however, likely that electrostatic effects are not the only determinants of the higher order assembly of AS brils. The presence of a disordered C-terminal region could also contribute to repulsion between brils as a result of entropic forces, such as those observed in colloidal suspensions stabilised through graed polymers; 63 indeed, it has been pointed out previously that the disordered regions of proteins in amyloid brils can behave like polymer brushes. 64,65 Overall, therefore, these observations conrm that C-terminal truncation of AS enhances the formation of higher order assemblies of AS brils at pH 6.5. This process is likely to reduce the accessibility of the bril ends for interaction with monomeric protein, and of the bril surfaces for binding to ThT molecules, thus providing a rationale for (i) the inability of Ctt AS seeds to initiate efficient aggregation (Fig. 2a) and (ii) the decrease in the ThT uorescence yield in the presence of Ctt AS brils (Fig. S4 †). Furthermore, the smaller degree of conversion of soluble protein molecules into amyloid brils during the time scale of our experiments can be explained through a signicant reduction in the kinetics of bril elongation due to the higher order assembly of the brils. This effect has the potential to slow down the aggregation process to such a large degree that it can appear already to have reached equilibrium during the timescale of the experimental measurements.  Solution pH differentially affects the ability of Ctt AS brils to proliferate through autocatalytic secondary nucleation We next investigated the effect of C-terminal truncations on the ability of AS brils to promote autocatalytic secondary nucleation. We have described in previous studies that the formation of new aggregates of AS(WT) by secondary nucleation at the surface of existing brils is very strongly dependent on the pH value of the solution. 28,29 The secondary nucleation rate of AS(WT) changes by several orders of magnitude between pH 6.0 and pH 5.5, an effect attributed to a change in the protonation state of the acidic residues in the C-terminal region of AS. 29 In the light of this conclusion, C-terminal truncations of AS could very signicantly inuence the rate of autocatalytic secondary nucleation. We therefore studied the aggregation behaviour of AS(WT), AS(1-119) and AS(1-103) at pH values ranging from 2 to 8 ( Fig. 5 and S8 †).
In our previous studies of AS(WT), we probed secondary nucleation by performing aggregation experiments in the presence of very low concentrations of seed brils. 29,66,67 Such experiments with the Ctt variants of AS are much more difficult to perform in a quantitative manner as a result of the tendency for higher order assembly of the Ctt AS brils described above, and so we used a different strategy. We incubated monomeric protein in uncoated polystyrene plates (also called "binding plates", see Experimental section for details), which were found to be able to initiate the aggregation of AS(WT). 26,28 Under these conditions, amyloid bril formation by AS is nucleated at the polymer surface. However, under quiescent conditions, where bril fragmentation as a pathway of autocatalytic bril ampli-cation 57 is not signicant, 29 aggregation can only be detected under conditions where secondary nucleation is able to amplify the small number of nuclei that form through heterogeneous primary nucleation. This concept is illustrated by the data for AS(WT) (Fig. 5a, S8a and b †), where we observed a sigmoidal increase in ThT uorescence at pH values ranging from 3.6 to 5.6. The upper bound of this range coincides with that of the conditions under which secondary nucleation is detectable in weakly seeded reactions. 29 We therefore used the observation of aggregation in polystyrene plates under quiescent conditions as indicative of a signicant contribution of secondary nucleation to the overall self-assembly process.
On this assumption, we studied the pH dependence of the aggregation of the Ctt AS species under these conditions and we found detectable aggregation between pH 4.2 and 7.0 for AS(1-119) and between pH 5.6 to 8.0 for AS(1-103) (Fig. 5b, c and S8c, d †). Outside these pH values, the formation of ThT-active species was not measurable (Fig. 5b, c and S8c, d †). Within the pH range where aggregation was observed, we found that the half time of the reaction, i.e. the time required for half of the soluble protein to be converted into aggregates, decreased signicantly as the solution pH approached the pI values (Fig. 5d). Moreover, the lag time of the reaction, i.e. the time at which a threshold fraction (we here used 0.5%) of the ThT uorescence at the plateau phase has been reached, was found to decrease with increasing concentrations of monomeric protein (Fig. S9 †).
These results suggest that the pH range where secondary nucleation contributes to amyloid formation by AS shis to higher values as the number of residues truncated from the Cterminal region of the protein is increased (Fig. 5e). The nding that C-terminal truncation has such signicant effects on the pH dependence of the secondary nucleation process of AS provides strong support for the hypothesis that this effect is principally determined by changes in ionisation of the Cterminal region. 29 Interestingly, C-terminal truncation does not abolish the strong pH-dependence of secondary nucleation, but rather signicantly shis the pH window within which it can be observed.
In order to conrm that the efficient proliferation of aggregates within the distinct pH-regions observed here is indeed due to the almost complete lack of electrostatic repulsion, we performed aggregation experiments in the same setup at varying salt concentrations, for each AS variant at the pH where aggregation was found to be most efficient (pH 5 for AS(WT), pH 6 for AS(1-119), pH 7 for AS(1-103), see Fig. S10 †). We found a very weak dependence of the lag times of the aggregation reaction on the ionic strength of the solution, therefore con-rming that the elimination of the majority of the electrostatic repulsion is a necessary requirement for proliferation through secondary nucleation.

C-terminal truncation affects the mechanism of lipid-induced aggregation of AS
In order to probe directly whether or not the de novo formation of amyloid brils is also affected by C-terminal truncations of AS, we performed aggregation experiments in the presence of SUV made from DMPS at pH 6.5 under quiescent conditions in PEG-ylated plates (also called "non-binding plates"), according to previously established protocols. 24 We have shown that for AS(WT) under these conditions, no secondary processes occur at appreciable rates, while at the same time the rate of primary nucleation is increased by several orders of magnitude compared to that in the absence of lipids. 24 Therefore, this system is well-suited to study the primary nucleation reaction associated with amyloid formation by AS.
We rst characterised the binding of AS(1-119) and AS(1-103) to the vesicles. Since AS adopts an a-helical structure upon membrane binding, we used CD to study the interaction between the Ctt variants and DMPS. The CD spectra of AS(1-119) and AS  in the presence of an excess of lipid vesicles are characterised by two minima, at 208 and 222 nm, as previously reported for AS(WT), 24 and are nearly superimposable on the spectrum of AS(WT) (Fig. S11a †). This observation suggests that the number of residues involved in the formation of the amphipathic a-helix is similar 68 for the three AS variants, a conclusion supported by an estimation using CDPro of the percentage of residues in a-helical structure: 80, 97 and 100% for AS(WT), AS(1-119), AS(1-103), respectively.
The binding curves of the AS variants to DMPS vesicles are all well described by a single step binding model 24 (Fig. S11b †); we found that, as with AS(WT), both AS(1-119) and AS(1-103) bind to DMPS vesicles with sub-micromolar affinity and that the stoichiometry (the number of lipid molecules per bound protein molecule) was 23 compared to 28 for AS(WT) (Fig. S11b †). This observation can be rationalised by the fact that the degree of steric and electrostatic repulsion among monomers on the surface of vesicles is likely to decrease as the length of the highly negatively charged C-terminus decreases.
We then studied the effects of the C-terminal truncations on the lipid-induced aggregation of AS by incubating each variant either at increasing concentrations of monomeric protein with a xed concentration of DMPS (Fig. 6a-c) or at a xed concentration of monomeric protein and increasing concentrations of DMPS ( Fig. 6d-f). As observed for AS(WT), 24 both AS(1-119) (Fig. 6d) and AS(1-103) (Fig. 6e) alone were not found to form detectable quantities of brils in the absence of DMPS vesicles under these experimental conditions, but the lipid vesicles were able to initiate the aggregation of both Ctt variants. The aggregation curves of AS(1-119) were characterised by the absence of a lag phase and by a sharp increase in uorescence intensity, the rate of which was dependent on the initial concentration of protein and that of DMPS ( Fig. 6a and  d). At the plateau phase, the concentration of insoluble AS(1-119) increased with the initial concentration of monomeric protein without reaching more than 20% of its value, suggesting that a large fraction of monomeric protein remains in solution at this stage (Fig. 6c). Increasing the initial concentration of DMPS also led to an increase in the concentration of insoluble AS(1-119) at the plateau phase, and the majority of monomers were found to convert to amyloid brils for concentration of DMPS of 500 mM (Fig. 6f). This aggregation behaviour is similar to that reported for AS(WT) 24 and can be rationalised through the cessation of both elongation and primary nucleation, as well as a negligible contribution of secondary processes that would be able to create new aggregation competent bril ends. 24 Since AS(1-119) appears to aggregate through the same mechanism as that of AS(WT), we analysed the early time points of the lipid-induced aggregation data using the one-step nucleation model, which describes well the lipid-induced aggregation of AS(WT) 24 (Fig. S12 †). We found that AS(1-119) aggregates an order of magnitude more rapidly than AS(WT) under these conditions (k n k + ¼ 3.01 AE 0.04 Â 10 À4 (AS(WT)) and 2.10 AE 0.03 Â 10 À3 (AS(1-119)) mol À1.2 s À2 ). The t to a single step model for the AS(1-119) kinetic data is less good than that of AS(WT) (Fig. S12 †). It could be improved by including a conversion step, representing an extension of the single step model that we have derived previously. 24 However, in order to be able to compare the rate constants directly, and to avoid over-tting, we decided to use the simpler model in both cases.
In the case of AS(1-103), we observed a different aggregation behaviour from that of AS(WT) and AS(1-119). First, the aggregation curves of AS(1-103) are characterised by a short lag phase followed by multiple increases and plateaus (Fig. 6b and e). Then, the concentrations of insoluble species at the nal plateau phase were found to be proportional to the initial concentration of monomeric AS(1-103) (Fig. 6c) but independent of the concentration of DMPS (Fig. 6f). These results suggest that all the soluble protein has been consumed at the plateau phase of the aggregation reaction of AS .
In order to obtain additional insights into the origin of the differences in the lipid-induced aggregation behaviour of AS(WT) and AS(1-119) on the one hand and AS(1-103) on the other, we characterised the morphology of the brils formed at different time points during the aggregation process using TEM (Fig. S13 †). At the plateau phase of AS(1-119), the majority of brils were found to be thin and curly, clearly similar to those observed for AS(WT). 24 We have previously suggested that these structures are protobrils that are kinetically trapped in an elongation incompetent state; we also observed that they can be converted into mature brils if mechanical forces, e.g. generated by sonication, are applied, 24 releasing new elongation competent bril ends. Interestingly, such protobrils were also detected at early times during the lipid-induced aggregation of AS(1-103) but they were no longer visible at the nal plateau phase, where the dominant species were observed to be straight and with the thickness characteristic of mature brils (Fig. S13 †).
The data shown in Fig. 5 indicate that at pH 6.5 in the absence of vesicles, secondary processes contribute signicantly to the overall aggregation reaction of AS(1-103) and AS(1-119), a result observed for AS(WT) at pH values below 5.5. 29 Our results therefore suggest that the lipid-induced protobrils formed by AS(1-103) are able to act as suitable templates for secondary nucleation. The nding that the lipid-induced pro-tobrils of AS(1-119) do not show secondary processes at pH 6.5 but that those produced by the polymer surface-induced process reveal the existence of such events, indicates that the surface properties of the brils nucleated in the presence of lipids or at the surface of plates have signicant differences. This conclusion is corroborated by the very different uorescence intensity associated with ThT binding that these two types of brils display (Fig. S14 †).

Conclusions
We have investigated in detail the effects that C-terminal truncations have on the overall mechanism of amyloid formation by AS. To that end, we have applied a strategy that enables us to study individual processes that constitute the overall aggregation mechanism, such as bril elongation, higher order assembly and autocatalytic proliferation, and the lipid-induced aggregation.
Overall our results show that the most signicant effect of the C-terminal truncations of the AS sequence is the dramatic change in the pH range within which autocatalytic secondary nucleation plays a role in the aggregation process. While the WT sequence displays secondary nucleation only at mildly acidic pH values, which are indeed encountered within a range of organelles, such as lysosomes and endosomes, the secondary nucleation pH range of the Ctt AS variants extends into the neutral region, therefore giving aggregates formed from these sequences the potential to proliferate within the cytosol (Table   S3 †). This insight is signicant in the light of the nding that considerable fractions of the aggregated forms of AS in vivo display C-terminal truncation and therefore this posttranslational modication may play a crucial role in the overall potential of AS to form aggregates that are able to proliferate exponentially and spread throughout the diseased brain.

Conflicts of interest
There are no conicts of interest to declare.