Atomic resolution map of the soluble amyloid beta assembly toxic surfaces† †Electronic supplementary information (ESI) available: Methods, 15N-DEST profiles, and additional statistical analyses. See DOI: 10.1039/c9sc01331h

Atomic resolution map of the soluble amyloid beta assembly (Aβn) “toxic surfaces” that facilitate the early pathogenic events in Alzheimer's disease (AD).


Introduction
While the etiology of Alzheimer's disease (AD) is complex and not fully understood, genetic mutations provide compelling evidence that the amyloid beta (Ab) peptide plays a critical role in AD pathogenesis. 1,2 Indeed, mutations in the genes encoding either the Ab progenitor (i.e. the amyloid precursor protein or APP) or the APP processing enzyme (presenilin 1 and 2 genes) are sufficient to cause AD. 1 Moreover, none of the familial AD mutations involve genes encoding for the tau protein. Instead, tau mutations enhance the deposition of neurobrillary tangles i.e. the other neuropathological hallmark of AD, but not amyloid plaques, and lead to different neuropathological disorders. 3 These genetic signatures coupled with the observation that Ab deposition precedes other biochemical and histopathological changes, including neurobrillary tangle formation, 4 provide evidence that tau aggregation occurs downstream to Ab aggregation. In addition, Ab clearance is controlled by one of the most signicant risk factors for late onset AD, i.e. APOE4. 5 Given the genetic link between Ab and AD, one of the main hypotheses proposed to explain AD pathogenesis is the amyloid cascade. The amyloid hypothesis posits that neuronal death in AD patients is associated with the increased production, self-association and accumulation of Ab in the brain. 2 Since it was originally postulated, the generality of the amyloid cascade hypothesis has been challenged because Ab plaque burden correlates poorly with cognitive dysfunction. 6 However, this inconsistency has been reconciled by considering that soluble oligomers and protobrils formed during the selfassociation cascade towards mature brils are neurotoxic 7 and better correlate with cognitive impairment in the early stages of AD. 8 Moreover, the neurotoxicity of Ab oligomers has been linked to tau hyperphosphorylation, 9 providing further evidence in support of the upstream role of soluble Ab assemblies in the AD pathogenesis cascade. 1 The central role of soluble Ab oligomers and protobrils in AD has prompted substantial efforts to identify the molecular determinants of neurotoxicity in soluble Ab assemblies (Ab n , where n represents the number of Ab molecules comprising the assembly). [10][11][12][13][14][15][16][17][18][19][20][21][22] Unfortunately, given the transient and heterogeneous nature of Ab intermediates, characterization of their structure and properties has been challenging. Despite these hurdles, it has been possible to delay the growth of aggregation intermediates to an extent sufficient to enable structural elucidation. For example, Ahmed et al. have shown that toxic Ab 42 oligomers stabilized through low temperature and salt conditions are largely disordered, but exhibit a turn conformation reminiscent of protobrils and brils. 20 In contrast, for the other major isoform of Ab, i.e. Ab 40 , toxic oligomers adopt parallel, in-register b-sheets. 21 While these studies have provided an initial framework to dene structural features of toxic Ab n , the location of the "toxic Ab n surfaces" remains unclear. Mapping such surface sites is critical as the exposure of toxic surfaces shared by multiple soluble Ab n species has been hypothesized to be one of the main causes of Ab n toxicity. 1,23 Exposure of these toxic surfaces is thought to facilitate interactions with multiple cellular components, including membranes, which underlie key pathogenic steps in the progression of AD. 1,22,[24][25][26] In fact, extracellular Ab oligomers are known to perturb biological and biomimetic membranes at multiple levels. The oligomers can (i) bind to membranes causing local perturbations, 19,27 (ii) form annular structures that insert into the membrane and affect ion homeostasis 16,18,19 and (iii) bind to membrane receptors altering signal transduction pathways. 28 Similar hypotheses have been proposed to explain the neurotoxicity of Ab protobrils, 17 although the latter have been shown to act also through detergent-like permeabilization and eventual fragmentation of the membrane. 19 While these results highlight critical aspects of Ab-membrane interactions, the "toxic surfaces" that enable key interactions with the membrane, as well as the underlying mechanism, remain elusive.
As a further step towards dissecting the molecular determinants of soluble Ab n toxicity and mapping the toxic Ab n surfaces, here we systematically investigate a library of Ab 40 assemblies sampling different degrees of cellular toxicity. To this end, we rst stabilized canonical, toxic Ab 40 assemblies through desalting and low temperature 29 and then treated them with a diverse set of catechins, ranging from (À)-epigallocatechin-3-gallate (EGCG), which remodels Ab into nontoxic structures, 30 to (À)-epicatechin (EC), which is expected to detoxify Ab only partially. We then proled our soluble Ab library through multiple complementary techniques with different degrees of spatial resolution, including extrinsic uorescence, electron microscopy, dynamic light scattering, wide-angle X-ray diffraction and NMR spectroscopy. Unlike previous attempts to dissect the toxicity determinants of Ab assemblies, 20,21 here we characterize representative soluble Ab assemblies from our library both in the absence and presence of model membranes.
The comparative analysis of our soluble Ab 40 library reveals a cluster of key toxicity determinants and the associated mechanism of action. We discovered that toxicity scales proportionally to the enhanced hydrophobic exposure of Ab 40 assemblies and their ability to interact with Ab monomers and cell membranes. The hydrophobic region spanning residues 17-28 is more accessible to monomer recognition in toxic Ab n relative to Ab n with reduced cellular toxicity. Moreover, whereas increased exposure of hydrophobic residues is required for toxicity, we nd that shielding of the highly charged Nterminus, i.e. residues < 12, from Ab monomer recognition enhances the toxicity of Ab n . These toxic Ab n surfaces are critical for the binding of Ab n to lipid membranes and for forming membrane-embedded b-sheet structures, which compromise the integrity of the cell membrane. The resulting model provides a foundation to start dening structure-toxicity relationships of Ab assemblies.

Results and discussion
An Ab 40 assembly library that samples a cytotoxicity gradient As a rst step towards dissecting the determinants of Ab 40 toxicity, we prepared a library of soluble Ab n spanning a cytotoxicity gradient. For this purpose, we incubated canonical (non-treated) Ab n with a collection of seven distinct catechins expected to remodel to varying extents the pre-existing soluble toxic Ab n into less toxic species 30-32 (ESI Fig. S1, † Methods). Out of this Ab n library, we selected a sub-set of representative Ab assemblies (i.e. those formed in the presence of the EC, (À)-epigallocatechin (EGC) and EGCG catechins) for toxicity proling in a human retinal pigment epithelial (RPE1) cell line. The state of the RPE1 cells was rst monitored by performing PrestoBlue assays, which rely on the reductive potential of the cell as a proxy of cellular viability. 33 Relative to mock (i.e. PBS delivery vehicle), canonical Ab n signicantly decrease cellular viability (Fig. 1a, black vs. grey). In contrast, Ab n formed in the presence of catechins are less effective in reducing cellular viability, in the order EC (Fig. 1a, green), EGC (Fig. 1a, yellow) and EGCG (Fig. 1a, maroon), for which no signicant difference is detected compared to mock (Table S1 †). Only negligible changes in cellular viability were observed for cells treated with catechins alone (Fig. 1a, dark green, orange and brown).
We also stained RPE1 cells with the necrotic cell marker propidium iodide (PI), which binds to DNA in cells with severely compromised membranes. 34 The RPE1 cells were also counterstained with the nuclear marker Hoechst 33342 35 to show that non-specic PI-staining is negligible under our conditions, as indicated by the purple vs. red uorescence for PI in merged vs. separate panels, respectively (Fig. 1b). Fluorescence microscopy images of RPE1 cells treated with canonical Ab n indicate prominent staining with PI ( Fig. 1b). In contrast, Ab n formed in the presence of catechins exhibit remarkably less PI staining (Fig. 1b), following the same EC < EGC < EGCG ranking as the cellular viability assay (Fig. 1a). Overall, these results suggest that the Ab assemblies in our library elicit different levels of cellular dysfunction and cell death. Hence, the comparative analysis of such Ab aggregates is anticipated to reveal key molecular determinants of soluble Ab toxicity.
The Ab assembly library spans a wide distribution of sizes, hydrophobic solvent exposures and cross b-sheet contents We rst evaluated how our catechin library remodels the distribution of Ab assemblies. For this purpose, the relative populations of the NMR visible low MW Ab species (e.g. monomers) were gauged through residual 1 H NMR intensities (Fig. 1c), while the NMR invisible Ab n were probed by dynamic light scattering (DLS) (Fig. 1d and e). While it is important to complement these data with size estimations through other means, such as TEM (vide infra), interestingly, we observed that all catechins in our library reduce the populations of both the Ab monomers (Fig. 1c) and the Ab assemblies at the opposite end of the molecular weight (MW) distribution ( Fig. 1d and e). These results suggest that the Ab species at the extremes of the probability distribution are converted by the catechins into Ab species with intermediate MW. However, the extent of this remodeling is markedly catechin-dependent with (À)-catechin-3-gallate (CG) leading to large reductions in both the monomer and high MW populations ( Fig. 1c-e) and methyl-3,4,5trihydroxybenzoate (MG) causing only marginal changes ( Fig. 1c-e). We also investigated the surface hydrophobicity of the Ab assemblies formed under our conditions, as exposed hydrophobic surfaces have been associated with toxicity for another amyloidogenic system. 36 The surface hydrophobicity of Ab n was probed through 8-anilino-1-naphthalenesulfonic acid (ANS) uorescence, which exhibits a characteristic blueshi and enhancement in uorescence intensity upon binding exposed hydrophobic sites. A substantial enhancement in ANS uorescence was observed for canonical Ab n ( Fig. 1f and g, black), whereas the extent of such enhancement is signicantly reduced for most catechin-treated Ab n ( Fig. 1f and g, coloured). Notably, the measurements of the catechin-treated Ab n surface hydrophobicity ( Fig. 1f and g) rank in the same order as the cell toxicities ( Fig. 1a), suggesting that exposed hydrophobic surfaces are a key determinant of Ab n toxicity.
Another unique signature of amyloids is the formation of extensive cross b-sheets, as reported by the uorescent dye Thioavin T (ThT). Canonical, toxic Ab n exhibit signicant ThT uorescence in comparison to catechin-remodeled Ab n (Fig. 1h). While the decreased ThT uorescence in the presence of EGCG is in agreement with previous observations, 30,37,38 our data on the extended catechin library reveal that other catechins also preserve the ability to destabilize intermolecular b-sheets and/or outcompete ThT. Hence, ThT-responsive b-amyloids do not appear to correlate with cytotoxicity as well as the observables reported above i.e. size and hydrophobic exposure. Indeed, solvent accessible hydrophobic moieties are one of the main drivers for Ab-membrane interactions, which in turn have been proposed as a key determinant of the cytotoxicity associated with Ab. 39 This hypothesis is supported by our propidium iodide results, which indicate that toxic Ab n severely compromise the integrity of cell membranes (Fig. 1b). To further corroborate this hypothesis, we evaluated the interactions between a representative subset of our Ab n library and biomimetic membranes (small unilamellar vesicles, SUVs).

Toxic Ab assemblies co-localize, bind and insert into biomimetics membranes
We proled the membrane interactions of selected Ab assemblies from our library that report on representative regions of our toxicity scale, i.e. the canonical as well as the EC-and EGCGremodeled Ab n (Fig. 1a). For this purpose, SUVs composed of a mixture of DOPE : DOPS : DOPC lipids were prepared with an effective size distribution ranging from $10-100 nm and an average diameter of $34 nm ( Fig. 2a and b). Prior to the addition of the Ab n library to the SUVs, we characterized the morphology of the Ab n by TEM to ensure that signicant catechin-induced remodeling occurs. Indeed, compared to canonical Ab n , which primarily adopt "worm-like" protobrils ( Fig. 2c, top le panel), we observed both spherical assemblies and amorphous aggregates in the presence of EGCG (Fig. 2c, top right panel). The latter of the two species has been reported to be an intermediate in the formation of the former. 23 In contrast, the EC-remodeled Ab n displays features of both canonical and EGCG-remodeled Ab n , albeit more closely resembling the canonical Ab n (Fig. 2c, top center panel). Having conrmed that catechin-induced remodeling of Ab n occurs, we then evaluated to what extent the Ab n library interacts with SUVs.
TEM images reveal that canonical Ab n signicantly colocalize with SUVs. For example, it is possible to observe select Ab n copositioned with the lipids (Fig. 2c, bottom le panel). Similar to the canonical Ab n , EC-remodeled Ab n are also somewhat colocalized with the SUVs (Fig. 2c, bottom center panel). However, in stark contrast to both the canonical and ECremodeled Ab n , the EGCG-remodeled Ab n are on average spatially distinct from the SUVs (Fig. 2c, bottom right panel).
To complement the TEM data on canonical vs. catechinremodeled Ab n -membrane interactions, we performed 15 Ntransverse relaxation (R 2 ), 1 H-based saturation transfer difference (STD) as well as 15 N-Dark State Exchange Saturation Transfer (DEST) NMR experiments, which collectively probe the interactions of Ab with high MW (HMW) species, including SUVs, Ab n and their complexes, through the lens of the NMR visible Ab monomers ( Fig. 3a-g). 29,[40][41][42][43][44][45][46][47][48] Upon addition of SUVs to the canonical Ab n , we observed marked enhancements in R 2 and STD ( Fig. 3a and b), consistent with the Ab n -membrane interactions revealed by TEM (Fig. 2c). The SUV-induced changes in R 2 and 1 H-based saturation transfer are more pronounced for the residues in the b1 (residues 12-24) and b2 regions (residues 30-40) than for the N-terminal moiety (residues < 12), indicating that the b1 and b2 segments serve as key hot-spots of the SUV-Ab interactions under our experimental conditions. This conclusion is independently conrmed by the comparative analysis of the 15 N-DEST data (Fig. 3g-m).
Residues in direct contact with the Ab n /SUV surface typically display an attenuation of the residual monomer DEST signal, leading to broadening of the residue-specic 15 N-DEST vs. offset prole relative to amino acids for which the monomer is disengaged from the Ab n /SUV surface. 31 Such broadening of the 15 N-DEST prole is quantitatively measured through the Q parameter at intermediate 15 N-continuous wave (CW) offsets, 40,49,50 as explained in the Methods. Consistent with the R 2 and STD data ( Fig. 3a and b), upon SUV addition to canonical Ab n , major DEST vs. offset prole broadening and corresponding Q enhancements are observed for the b1 and b2 regions ( Fig. 3c-g and k; ESI Fig. S3 †). A similar observation applies to the addition of SUVs to EC-remodeled Ab, which on average display a pattern comparable to canonical Ab n (Fig. 3h and l vs.  Fig. 3g and k; ESI Fig. S4 †). Conversely, the EGCG-remodeled Ab do not exhibit signicant b1 and b2 enhancements as compared to canonical and EC-remodeled Ab (Fig. 3i and m; ESI Fig. S5 †), in excellent agreement with the TEM observations. While the combination of our TEM and 15 N-based NMR experiments reveal key differences in Ab-membrane interactions between the less toxic EGCG-remodeled Ab and the more toxic canonical and EC-remodeled Ab, they do not provide direct insight about whether Ab n inserts into the membrane and about the structural features of membrane-embedded Ab n . To this end, we conducted wide-angle X-ray diffraction (WAXD) experiments in the presence of model membranes for Ab assemblies at representative regions of our toxicity scale (Fig. 2d-l).
The WAXD two-dimensional intensity maps (Fig. 2e) were modeled with a series of Lorentzian ts (Methods) to derive structural features both in-plane (q k , Fig. 2f-k) and out-of-plane (q z , Fig. 2l) of the membrane. For the lipid sample in the absence of Ab n , in-plane and out-of-plane Bragg peaks were observed at 1.41Å À1 (Fig. 2f) and 0.17Å À1 (Fig. 2l, black), respectively, corresponding to the formation of bilayer stacks with an effective bilayer width of 38.7Å and a 5.1Å spacing between individual lipids (Fig. 2d). Addition of canonical Ab n to these lipid bilayers results in additional in-plane features at 1.32 A À1 (Fig. 2g, blue) and 0.76Å À1 (Fig. 2k, red), indicating the presence of membrane-embedded Ab n adopting laminated bsheets with 5.5Å spacing between adjacent b-strands and 9.5Å between b-sheet layers (Fig. 2d). Interestingly, we observe an additional peak at 1.51Å À1 (Fig. 2g, cyan) corresponding to highly ordered lipids likely in the regions interfacing with the embedded Ab n . Moreover, an out-of-plane diffraction pattern is observed at $0.12Å À1 (Fig. 2l, red) consistent with the presence of Ab not embedded into the bilayer (Fig. 2d).
Compared to canonical Ab n , the EC-and EGCG-remodeled Ab n still preserve extended b-sheets in the membrane ( Fig. 2h  and i, blue), although the relative amounts are decreased in the presence of EC and EGCG, in that order (Fig. 2j). In contrast, neither of the catechin-remodelled Ab n exhibit packing of bsheet layers (Fig. 2k, green and blue), in agreement with our ThT data (Fig. 2h). Overall, these ndings suggest that the toxic Ab n formed under our conditions colocalize, interact and insert into lipid membranes wherein they adopt b-sheet structures. To identify the toxic Ab n surfaces that facilitate these multivalent interactions with the membrane, we comparatively examined the 15 N-DEST differences between canonical, EC-and EGCGremodeled Ab n in the presence of model membranes (Fig. 3n,  ESI Fig. S2 †).
Toxic vs. non-toxic Ab assemblies in the membrane environment exhibit marked differences in Ab-recognition proles To focus on the effects of the catechins, the canonical Ab n DEST prole (ESI Fig. S2b †) was subtracted from the catechinremodeled Ab n DEST proles (ESI Fig. S2c and d †). Since all proles in ESI Fig. S2b-d † were recorded in the presence of SUVs, the resulting DEST differences (Fig. 3n) report primarily on the catechin-induced remodeling of Ab monomer-Ab n contacts. Specically, the EGCG-remodeled vs. canonical Ab n 15 N-Q prole differences (D EGCG Q) show signicant decreases in Q in the two b-strand regions typically observed in Ab pro-tobrils (Fig. 3n, darkblue). These losses are consistent with the Ab monomers being less engaged with the Ab n surface at the two b-strand sites in the presence of EGCG. However, the EGCGinduced disengagement detected for the b1 and b2 regions does not extend to the N-terminal segment, for which a signicant enhancement in direct contacts is observed (Fig. 3n,  darkblue). A similar N-terminal Q DEST enhancement is observed also upon EC addition (Fig. 3n, lightblue), albeit with reduced magnitude (Fig. 3n, light vs. darkblue). Likewise, in the b1 region the EC-remodeled Ab n show Q losses with a reduced extent compared to the EGCG-remodeled Ab n (Fig. 3n, light vs. darkblue). However, the DEST pattern observed for the Nterminal and b1 regions does not extend to the b2 segment, for which EC and EGCG result in opposite Q changes (Fig. 3n,  light vs. darkblue). These ndings imply that exposure of the hydrophobic b1-turn region and concomitant shielding of the N-terminus are two key structural transitions intimately linked to toxicity, as these toxic surfaces modulate interactions with the membrane.

Selection of molecular determinants of Ab n toxicity
In order to systematically isolate the Ab n features relevant for toxicity, we identied groups of coupled Ab n observables by relying on the data correlation matrix (Fig. 4a), whose elements represent the absolute Pearson's correlation coefficients (|r|) between each pair of Ab n observables (ESI †). Through agglomerative clustering of the correlation matrix, we then built a dendrogram that partitions the Ab n observables into ve distinct clusters (Fig. 4b). The largest cluster, denoted as cluster 1, includes the D Cat Q i values for residues in the 3-28 region as well as three low resolution observables, i.e. the membraneembedded b-sheet, the size and the surface hydrophobicity. Since these measurables rank similarly to the relative toxicities ( Fig. 1), we hypothesized that cluster 1 denes key molecular determinants of Ab n toxicity. This hypothesis is conrmed by two independent lines of evidence.
First, if we re-compute the correlation matrix and agglomerative clustering aer including the relative toxicities (Fig. 1a), we nd that the toxicity partitions within cluster 1 (ESI Fig. S6 †), conrming that the observables in this cluster scale with Ab n toxicity. Second, in the D EGCG Q i vs. D EC Q i plot (Fig. 4c), the cluster 1 residues fall at or near the region expected to scale with the relative EC vs. EGCG cell viability (CV) data, dened as (CV Ab 40 +EC À CV Ab 40 )/(CV Ab 40 +EGCG À CV Ab 40 ) ¼ 0.42 AE 0.05 (shaded blue area, Fig. 4c). The linear regression of D EGCG Q i vs. D EC Q i for cluster 1 is in fact in excellent agreement with the value expected based on the relative cellular viability (dashed blue line with slope of 0.42 AE 0.02 and correlation coefficient of 0.98; Fig. 4c). Hence, we conclude that cluster 1 (blue dendrogram in Fig. 4b and blue circles in Fig. 4c) is relevant for the toxicity of Ab n .
To gain further insight on the signicance of the D EGCG Q i vs. D EC Q i plot and independently corroborate the residue clusters obtained through the agglomerative clustering analysis, we also performed Singular Value Decomposition (SVD) of the data in Fig. 4c. The SVD analysis reveals that the rst principal component (dashed black line, Fig. 4c), which accounts for 88% of the total variance, not only resides within the range expected to scale with the relative cellular viability (i.e. within the shaded blue area in Fig. 4c), but also aligns with the residues for cluster 1. Interestingly, the SVD reveals that cluster 1 (blue circles, Fig. 4c) is composed of two distinct sub-sets that are mostly conned at opposite extremes of PC1, between the 1s and 2s ellipsoids (Fig. 4c). The sub-set with positive PC1 components (dark blue circles) represents the N-terminal residues that become engaged in monomer recognition, as probed by DEST, when cellular viability is enhanced. On the contrary, the cluster 1 sub-set with negative PC1 scores (light blue circles) arises from the b1-turn region residues that become engaged when cellular viability decreases.
In stark contrast to cluster 1, the other clusters obtained from the agglomerative clustering analysis (Fig. 4b, black, green, red and orange circles) fall outside the range expected to scale with cellular viability (blue shaded area, Fig. 4c) and exhibit components along PC2 that are overall higher than those observed for cluster 1 (Fig. 4c). In conclusion, the combined analyses of the correlation matrix, agglomerative clustering and SVD consistently identify the constituents of cluster 1, i.e. surface hydrophobicity, size, membraneembedded b-sheets, N-terminal residue disengagement and b1-turn region engagement, as key molecular determinants of Ab n toxicity.
In order to verify the predictive power of the correlation between Ab n toxicity and cluster 1, we measured the relative toxicities for the Ab assemblies not included in Fig. 1a and we compared them to those predicted by our model (Fig. 4; ESI  Fig. S7 †). These Ab n toxicities were not used to train our model and hence provide a critical test of its prognostic capacity. As seen in ESI Fig. S7d, † a strong linear correlation is observed between the predicted and observed toxicities (r $ 0.94), with a slope within error to one, thus validating the predictive power of our model.
In summary, our investigation of the Ab n library through the comparative analysis of 15 N-R 2 and DEST NMR combined with WAXD, TEM, DLS and extrinsic uorescence reveals key structural differences that distinguish toxic vs. non-toxic Ab assemblies. The integrated analyses of our data through agglomerative clustering and SVD consistently identify a cluster of molecular attributes unique to toxic Ab n (Fig. 4b, cluster 1), including surface hydrophobicity, size, membrane-embedded b-sheets, shielding of the N-terminus and simultaneous exposure of the b1-turn region to Ab monomers, as probed through DEST NMR.
Our data shows that toxic Ab n exhibit solvent exposed hydrophobic sites accessible to ANS binding. While the relationship between surface hydrophobicity and toxicity has been observed previously for several protein systems such as the Type A/B HypF-N assemblies, 51,52 the A + /A À Ab 42 oligomer pair, 53 the sup35p oligomer pair, 54 and others, 55 here we not only systematically conrm this association for the Ab system using a library of Ab assemblies, but we also propose an unprecedented mechanism of Ab n toxicity probed at multiple degrees of resolution. Such mechanism reveals how hydrophobic exposure relates to Ab-membrane interactions and Ab monomer recognition. The combination of our TEM, DLS and 15 N-DEST and R 2 Fig. 5 Proposed model for the molecular determinants of Ab assembly toxicity. (a) Toxic Ab n (canonical Ab n ) exhibit significant solvent exposure of hydrophobic surfaces (yellow glow surrounding Ab n ). Exposed hydrophobic surfaces facilitate the colocalization, interaction and subsequent insertion of Ab n into the membrane. (b) Membrane-embedded Ab n adopt both laminated and non-laminated b-sheets, indicating that under our experimental conditions the non-laminated b-sheet signature is the minimum structural feature required for membrane insertion and induction of toxicity. (c) Toxic vs. non-toxic Ab n exhibit unique regiospecific differences in the recognition of Ab monomers within a membrane environment. Relative to canonical Ab n (black), EC-(green) and EGCG-remodeled Ab n (maroon) exhibit progressive engagement of contacts with Ab monomers at the N-terminus and disengagement at the b1-turn region, following the same ranking as their measured toxicities. In contrast, for the b2 region no correlation is observed between toxicity and Ab n monomer recognition. Relevant experimental techniques are indicated in parenthesis. (d) Mapping on the structure of Ab 40 fibrils 57 (PDB code: 2LMN) the Ab residues in cluster 1 (Fig. 4b and c). The N-terminal and b1-turn residues that correlate with toxicity (blue) are found in the external regions of the Ab fibril structure. In contrast, b2 is involved in the lamination of multiple b-sheet layers and is largely inaccessible (Table S2 †), explaining its ancillary role in toxicity. data collectively shows that Ab n with greater surface hydrophobicity e.g. canonical and EC-remodeled Ab n colocalize and interact with the membrane surface more effectively than the less toxic Ab n with less exposed hydrophobic sites e.g. the EGCGremodeled Ab n (Fig. 5a).
The surface hydrophobicity-mediated interactions with the membrane are not limited to the membrane surface, as our WAXD data show that canonical and EC-remodeled Ab n exhibit signicant populations of b-sheets embedded in the membrane compared to EGCG-remodeled Ab n . The functional effect of the membrane-embedded b-sheets is recapitulated by our propidium iodide-based assay, which indicates that canonical Ab n signicantly enhance the permeability of the cell membrane compared to the less toxic Ab n formed in the presence of EGCG.
Notably, we also found that cross-b-sheet structures are dispensable for membrane insertion, as only canonical Ab n exhibit cross lamination of b-sheet layers, whereas ECremodeled Ab n, with comparable levels of membraneembedded b-sheets, exhibit considerably reduced cross lamination, similar to EGCG remodeled Ab n ( Fig. 2k and 5b). The lack of correlation between toxicity and b-sheet crosslamination is also consistent with the variability in sheet-tosheet pairing angles reported for oligomers of model amyloidogenic sequences stabilized by macrocyclic peptides. 56 The correlation and SVD analyses also identify a cluster of residues conned to the N-terminus and b1-loop region that are key to the regulation of Ab n toxicity ( Fig. 4b and c, cluster 1). The probability distribution of contacts between Ab monomers and the Ab n /SUVs surface is markedly enhanced in the b1-loop region (residues [17][18][19][20][21][22][23][24][25][26][27][28] and concomitantly reduced at the Nterminal segment (residues 3-10) as the Ab n toxicity increases ( Fig. 3n and 5c, green vs. maroon arrows). Interestingly, an unexpected decorrelation with toxicity is observed at the b2 region (residues 30-40) (clusters 2 and 4), for which the ECremodeled Ab n , with intermediate toxicity, exhibits a further enhancement in contacts relative to the canonical Ab n (Fig. 3n and 5c, green), in stark contrast to the reduction observed for EGCG-remodeled Ab n ( Fig. 3n and 5c, green vs. maroon arrows).
Notably, the N-terminus and b1-loop Ab regions identied by the correlation and SVD analyses to be toxicity determinants (Fig. 4c, cluster 1) are located at the external surface of the Ab 40 bril structure (Fig. 5d, blue surfaces). Furthermore, most familial AD mutations (English, Tottori, Iowa, Arctic, Dutch and Italian) that alter the biophysical properties of Ab are observed in the N-terminal and b1 regions. 1,58 Conversely, the b2 region not identied by SVD as linked to toxicity, is inaccessible to the environment (Table S2 †) and is found embedded into the structural core of the bril, where it is involved in the cross lamination of multiple b-sheet layers (Fig. 5d, grey cartoon). These observations agree with our WAXD and ThT data, consistently pointing to b-sheet lamination as accessory to toxicity induction.

Conclusions
Overall, our data indicate that Ab n toxicity is regulated by the solvent exposure of hydrophobic surfaces, wherein the hydrophobic b1-turn region is more accessible to monomer/ SUV recognition, while the highly charged N-terminus is shielded from such recognition. In comparison, the role of b2 appears to be largely ancillary. These toxic surfaces enhance the colocalization, contacts and subsequent insertion of b-sheet rich Ab n into the membrane, leading to compromised membrane stability. Moreover, the proposed model is able to predict relative toxicities solely based on low-resolution measurements, such as size and surface hydrophobicity. Modulation of these properties through small-molecule treatment can be utilized as an effective strategy to reduce the toxicity associated with soluble Ab assemblies. In addition, soluble oligomers of amyloidogenic peptides with different sequences have been suggested to share a common conformation, 59 and Ab is not only relevant for dominantly inherited AD, but also serves as a model system for a broad-range of amyloid disorders. Hence, the cluster of molecular attributes identied here to correlate with toxicity may be transferrable to other amyloidogenic systems.

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