A β-barrel-like tetramer formed by a β-hairpin derived from Aβ

β-Hairpins formed by the β-amyloid peptide Aβ are building blocks of Aβ oligomers. Three different alignments of β-hairpins have been observed in the structures of Aβ oligomers or fibrils. Differences in β-hairpin alignment likely contribute to the heterogeneity of Aβ oligomers and thus impede their study at high-resolution. Here, we designed, synthesized, and studied a series of β-hairpin peptides derived from Aβ12–40 in one of these three alignments and investigated their solution-phase assembly and folding. These assays reveal the formation of tetramers and octamers that are stabilized by intermolecular hydrogen bonding interactions between Aβ residues 12–14 and 38–40 as part of an extended β-hairpin conformation. X-ray crystallographic studies of one peptide from this series reveal the formation of β-barrel-like tetramers and octamers that are stabilized by edge-to-edge hydrogen bonding and hydrophobic packing. Dye-leakage and caspase 3/7 activation assays using tetramer and octamer forming peptides from this series reveal membrane-damaging and apoptotic properties. A molecular dynamics simulation of the β-barrel-like tetramer embedded in a lipid bilayer shows membrane disruption and water permeation. The tetramers and octamers described herein provide additional models of how Aβ may assemble into oligomers and supports the hypothesis that β-hairpin alignment and topology may contribute directly to oligomer heterogeneity.


Introduction
The formation and biological activity of b-amyloid oligomers are central events in the pathogenesis and progression of Alzheimer's disease.Dimers of Ab isolated from Alzheimer's brains have been shown to disrupt neuritic integrity in vitro, and in a separate study impair synaptic structure and function in vivo. 1,2Synthetic trimers of peptides derived from Ab have been used to raise antibodies that recognize pathological features in tissues isolated from Alzheimer's brains. 3Tetramers and octamers of Ab have demonstrated pore-forming activity against lipid-bilayers. 4Photo-induced crosslinking studies of Ab 42 have revealed the formation of pentamers and hexamers, while ion mobility-mass spectrometry studies have shown that Ab 42 forms b-barrel shaped hexamers in the presence of membrane mimetic micelles. 5,6Understanding the biophysical and biological activity of Ab oligomers is key to understanding the molecular basis of neurodegeneration and cognitive decline in Alzheimer's disease.
3][14][15][16] To overcome these difficulties fragment-based investigations, covalentstabilization, synthetic Ab oligomer homologues, and in silico modeling have emerged as tools to aid in the study of more uniform and homogenous models of Ab oligomers. 17A substantial body of evidence has emerged from these investigations, identifying b-hairpins as a key structural element of Ab oligomers.
The NMR-based structure of a tetramer formed by full-length Ab 42 reported by Carulla and coworkers is the only atomicresolution structure of an Ab oligomer that has been deposited in the Protein Data Bank, (PDB 6RHY). 4The tetramer comprises a six-stranded antiparallel b-sheet, with two b-hairpins of Ab 42 that ank two antiparallel b-strands of Ab 42 (ESI Fig. S1a †).
Additional solution-phase studies of this tetramer also provide evidence for the formation of an octamer.Using molecular dynamics (MD) simulations, Carulla and coworkers propose a model in which this tetramer, as well as the octamer, can act to disrupt a lipid membrane and facilitate water permeation.Collectively, these studies have brought into sharp relief the importance of b-hairpins in the structures of Ab oligomers.
9][20][21][22] In 2008, Härd and Hoyer reported the NMR structure of a monomer of Ab 40 adopting a b-hairpin conformation when sequestered and stabilized by an affibody. 19Härd and coworkers subsequently stabilized this Ab b-hairpin using an intermolecular disuldebridge and found that the stabilized b-hairpin formed oligomers that mimicked some of the properties of oligomers formed by unmodied Ab. 20 NMR spectroscopic studies revealed that related disulde-stabilized b-hairpins derived from Ab 16-42 assembled to form a barrel-shaped hexamer stabilized by hydrophobic packing and edge-to-edge hydrogen bonding between b-hairpins (ESI Fig. S1b †). 21ycko and coworkers recently reported a b-hairpin as a component in the structure of an atypical Ab 40 bril structure, in which the characteristic core of parallel in-register b-sheets is coated by an outer layer of b-hairpins formed by Ab (ESI Fig. S1c †). 23A model of the b-hairpin that ts the cryo-EM and NMR spectroscopic data shows residues 16-22 and 30-36 hydrogen bonding to form a b-sheet, with the intervening residues 23-29 forming a loop (Fig. 1a).
The Ab b-hairpins reported by Carulla and coworkers, Härd and Hoyer and coworkers, and Tycko and coworkers all differ in the alignment of their b-strands (Fig. 1a). 4,19,23In the tetramer reported by Carulla and coworkers, b-strands comprising residues 9-21 and 28-40 hydrogen bond to form an antiparallel b-sheet, with residues 22-27 forming a loop.In the barrel-shaped hexamer reported Härd and coworkers, b-strands comprising residues 17-22 and 30-36 hydrogen bond to form an antiparallel b-sheet, with residues 23-29 forming a loop.These differences in b-strand alignment alter the overall topology of the b-hairpins by shiing residue pairings across the b-strands, the hydrophobicity of the bhairpin surfaces, and the size of the loop segments between the bstrands.In the b-hairpin reported by Carulla and coworkers, Ile 32 is across from Phe 19 ; in the b-hairpin reported by Tycko and coworkers, Gly 33 is across from Phe 19 ; and in the b-hairpin reported by Härd and Hoyer and coworkers, Val 34 is across from Phe 19 (Fig. 1a).These changes in alignment and topology may contribute to the immense variation and heterogeneity observed in the assembly and structures of Ab brils and oligomers.
In the current study, we set out to explore oligomers formed by b-hairpins in the alignment reported by Tycko and coworkers by designing, synthesizing, and studying a series of b-hairpin peptides derived from Ab 12-40 . 17,24The structures of these peptides are illustrated in Fig. 1b and c.Four of these peptides (1a, 1d, 1h, and 2a) assemble to form octamers in SDS-PAGE.Xray crystallographic studies of peptide 2a reveal a hitherto unprecedented b-barrel-like tetramer composed of b-hairpins.The crystallographic tetramers concatenate within the crystal lattice to create an octamer, and thus suggest a structural model for the octamers observed in SDS-PAGE.

Design of peptides 1a-i and 2a-d
We designed peptide 1a to fold into an Ab b-hairpin in the alignment reported by Tycko and coworkers to probe the assembly of Ab b-hairpins into oligomers.6][27][28][29][30][31][32][33] We designed homologous peptides 1b-i and 2a-d to further explore the effects of the N-and C-terminal residues 12-14 and 38-40, the Ab 23-29 loop, and additional d Orn turn units and disulde bridges on folding and assembly.

Oligomerization of peptides 1a-h and 2a-d
The formation of oligomers that can be observed in SDS-PAGE is a hallmark of Ab. 7,[34][35][36][37] Peptide 1a (2.5 kDa) runs as an oligomer in SDS-PAGE, migrating at a molecular weight consistent with that of an octamer (ca.20 kDa; Fig. 2a).The octamer band streaks downward from below the 26 kDa ladder band, suggesting that the octamer is in equilibrium with lower molecular weight species.Unlike the oligomer formed by peptide 1a, the oligomers formed by full-length, unmodied Ab are heterogeneous in size and typically display a substantial band for the monomer (Fig. 2a). 5,34,36The formation of a well-dened oligomer band by peptide 1a suggests that this constrained Ab bhairpin peptide forms a well-dened supramolecular assembly in the membrane-like environment provided by the amphiphilic SDS molecules.
To better understand the assembly of the putative octamer, we prepared and studied peptides 1b-h (Fig. 1c).Peptide 1b (2.5 kDa) lacks a cross-strand disulde bridge and instead contains the native Gln 15 and Gly 37 residues.Peptide 1b does not assemble in SDS-PAGE, migrating just below the 4.6 kDa ladder band (Fig. 2a).Peptide 1c (1.8 kDa) maintains the cross-strand disulde bridge but lacks the N-and C-terminal residues 12-14 and 38-40.Peptide 1c does not assemble, migrating as a downward-streaking band from the 4.6 kDa ladder band.In contrast to peptide 1c, peptide 1d (2.3 kDa) lacks only the Cterminal residues 38-40 and does assemble.Like peptide 1a, peptide 1d migrates as a downward-streaking band from below the just 26 kDa ladder band.The band formed by peptide 1d suggests an octamer in equilibrium with lower molecular weight species.Peptide 1e (2.1 kDa) lacks only the N-terminal residues 12-14 but does not assemble.Instead, peptide 1e migrates as an upward-streaking band from just below the 4.6 kDa ladder band.
The lack of oligomer formation by peptide 1b reveals that the cross-strand disulde bridge near the N-and C-terminal residues is necessary for assembly of the putative octamer.It has been noted that SDS is known to contribute to the assembly of small Ab oligomers. 38The differences in the oligomerization of peptides 1a and 1b suggests that stabilization of the b-hairpin conformation by the disulde bridge, rather than SDS, induces the formation of the putative octamer.The assembly of peptide 1d, in contrast to peptides 1c and 1e, is surprising and suggests that residues 12-14 participate in intermolecular interactions crucial for assembly of the putative octamer.
To further examine the role of interactions between the Nand C-terminal residues of peptide 1a, 12-14 and 38-40, in the assembly of the octamer, we prepared and studied peptide 1f.In peptide 1f, an N-methyl group on Gly 38 is positioned to disrupt hydrogen bonding interactions between residues 12-14 and 38-40.In contrast to peptide 1a, peptide 1f (2.5 kDa) does not assemble as an octamer, but instead migrates as a band between the 10 kDa and 4.6 kDa ladder bands that streaks downward.This result suggests that residues 12-14 and 38-40 hydrogen bond together as part of an extended b-hairpin in the octamer formed by peptide 1a.
To examine whether the putative octamer formed by peptide 1a can accommodate residues 23-29 as a loop, and to better mimic endogenous Ab b-hairpins and Ab oligomers, we prepared and studied peptides 1g and 1h.In peptide 1g a loop comprising residues 23-29 replaces the d Orn turn unit.In contrast to peptide 1a, peptide 1g (3.0 kDa) does not assemble and instead migrates just above the 4.6 kDa ladder band.Peptide 1h is a homologue of peptide 1g, that incorporates an additional cross-strand disulde bridge replacing Asp 23 and Gly 29 to fortify the b-hairpin conformation of the peptide.Peptide 1h (3.0 kDa) migrates as two bands, a lower molecular weight band consistent with a tetramer and a higher molecular weight band consistent with an octamer.The tetramer band migrates between the 10 and 17 kDa ladder bands and is much greater in intensity than the octamer band, which migrates just below the 26 kDa ladder band.The difference in intensity between the tetramer and octamer bands suggest that peptide 1h favors assembly of the tetramer.Thus, it appears that the octamer formed by peptide 1a can accommodate the Ab 23-29 loop, but that the loop destabilizes the b-hairpin conformation required for assembly-unless an additional stabilizing disul-de bridge is present, as in peptide 1h.
Peptide 2a is a macrocyclic homologue of peptide 1a with a second d Orn turn unit connecting the N-and C-terminal residues 12 and 40.Like peptide 1h, peptide 2a (2.6 kDa) migrates as two oligomers, a lower molecular weight oligomer consistent with a tetramer and a higher molecular weight oligomer consistent with an octamer.The tetramer band migrates just above the 10 kDa ladder band, and the octamer band migrates between the 17 and 26 kDa ladder bands.Both bands are equal in their intensity, with the octamer band streaking downward toward the tetramer band, suggesting that the two species are in equilibrium with each other.Peptide 2b is a homologue of peptide 2a that lacks the cross-strand disulde bridge and does not assemble, instead migrating just below the 4.6 kDa ladder band.
Collectively, the SDS-PAGE studies of peptides 1b-1h and 2a-2b highlight key factors in the formation of an octamer by peptide 1a.The N-terminal residues 12-14 form critical intermolecular contacts in the octamer.The formation of both tetramers and octamers by peptides 1h and 2a indicates that the tetramers and octamers are in equilibrium and suggests that the tetramers may be components of the octamers, for these peptides as well as for peptide 1a.Peptides 1h and 2a bear additional stabilizing constraints not present in peptide 1aa second disulde bridge and a second d Orn turn unit respectively.Rather than stabilizing the octamer, these additional constraints destabilize the octamer and promote the formation of a tetramer, which appears to be a subunit of the octamer.The lack of assembly by peptide 2b, despite the second d Orn turn unit, suggests that the cross-strand disulde bridge replacing Gln 15 and Gly 37 is essential for octamer assembly.

Folding of peptides 1a-h and 2a-b
The CD spectrum of peptide 1a is similar to that of a b-sheet, suggesting that the peptide folds as designed into a b-hairpin (Fig. 2b).The CD spectrum of peptide 1a displays a strong negative band centered at ca. 212 nm, with increasing ellipticity at lower wavelengths that reaches a maximum at ca. 198 nm before decreasing once again.The disulde bridge in peptide 1a appears to be essential for folding-the CD spectrum of peptide 1b indicates that it does not fold and that a random coil conformation predominates.Peptide 1b displays a strong negative band centered at ca. 198 nm and a weak maximum at ca. 220 nm (Fig. 2b).The CD spectra of peptides 1c-f and 1h show that these peptides also fold to adopt b-hairpin-like conformations.
Differences between the CD spectra of peptides 1a and 1g reveal that replacing the d Orn turn unit with Ab residues 23-29 abrogates b-hairpin folding (Fig. 2c).The CD spectrum of peptide 1g displays a broad and shallow negative band centered at ca. 204 nm, which suggests a random-coil-like conformation.Addition of a disulde linkage between residues 23 and 29 partially restores folding.Thus peptide 1h displays a negative band centered at ca. 212 nm with increasing ellipticity at lower wavelengths.
The CD spectra of peptides 2a and 2b suggest that incorporation of a second d Orn turn unit connecting residues 12 and 40 does not substantially affect folding (Fig. 2c).Peptide 2a displays a strong negative band centered at ca. 214 nm with increasing ellipticity at lower wavelengths, similar to peptide 1a, reecting a b-hairpin-like conformation.Peptide 2b displays a strong negative band centered at ca. 200 nm, similar to peptide 1b, indicating a random coil conformation.Thus, the constraint of peptide 1b into a macrocycle but without a disul-de bridge is not sufficient to induce b-hairpin folding.
Correlation of these CD studies with the SDS-PAGE studies suggests that folding is necessary for assembly, but that not all folded b-hairpin peptides can assemble.Key residues and contacts also appear to be necessary to achieve the putative octamer that is observed in SDS-PAGE for peptides 1a, 1d, 1h, and 2a.

X-ray crystallographic and REMD studies of peptide 2a
To gain further insights into the structures of the oligomers observed in SDS-PAGE, we grew crystals of peptide 2a and performed X-ray crystallography.None of the other peptides afforded crystals.The X-ray crystallographic structure of peptide 2a reveals that the peptide assembles to form antiparallel b-sheet dimers that further assemble in a face-to-back fashion to form concatenated b-barrel-like tetramers (Fig. 3, PDB 7RTZ).The core of each b-barrel-like tetramer is lined with hydrophobic residues (Cys 15 , Val 18 , Phe 20 , Ile 32 , Ile 34 , Met 35 , Val 36 and Cys 37 ), which pack against each other while creating a pore through the center, ca.7.5 Å in diameter (ESI Fig. S3b †).ESI Table S1 † summarizes the crystallographic properties, conditions, data collection, and model renement statistics for peptide 2a.
The b-hairpin subunits comprise residues 12-22 and 30-40 and include a disulde bridge that replaces Gln 15 and Gly 37 (ESI Fig. S3a †).The b-hairpin subunits assemble in pairs to form the antiparallel b-sheet dimers (ESI Fig. S3b †), which are stabilized by two pairs of intermolecular hydrogen bonds between Met 35 and Cys 37 .The two dimers further assemble through parallel bsheet interactions between His 14 and Phe 19 to form the eightstranded b-barrel-like tetramer (Fig. 3a-c).Molecular modeling suggests that full-length Ab can assemble in the same fashion as peptide 2a to form a b-barrel-like tetramer (Fig. 3d).Thus, we observed that Ab 9-42 could form a b-barrel-like tetramer without steric clashes when modeled into the crystallographic coordinates of the tetramer using replica exchange molecular dynamics (REMD) simulations in implicit solvent to generate realistic conformations of the loops and N-and Cterminal regions.
In the crystal lattice, the b-barrel-like tetramers link together to form concatenated networks of b-barrels running the length of the lattice, with the interface between each tetramer also constituting a b-barrel-like tetramer (Fig. 4).Thus, two tetramers may be thought of as composing an octamer consisting of three linked b-barrels.The two tetramers that make up the octamer are parallel to each other, and the tetramer formed at their interface is perpendicular but otherwise identical.Larger oligomers composed of more tetramers can also be envisioned.The crystallographically observed tetramers and octamers may explain the structures of the tetramers and octamers observed in SDS-PAGE for peptides 1a, 1d, 1h, and 2a, with each tetramer consisting of a single b-barrel and two tetramers further assembling to form an octamer consisting of three concatenated b-barrels.

Correlating the crystallographic tetramer and octamer with the oligomers observed in SDS-PAGE
To better understand the tetramers and octamers observed in SDS-PAGE and to correlate these species with the crystallographically observed tetramers and octamers formed by peptide 2a, we performed TCEP reduction, additional N-methylation, and disulde crosslinking experiments (Fig. 5a and b). 39eduction of the disulde bonds of peptides 1a and 2a with tris(2-carboxyethyl)phosphine (TCEP) disrupts octamer formation in SDS-PAGE.Treatment of peptide 1a with TCEP results in a streaky band that is similar in position to that of peptide 1b, which does not assemble (Fig. 5a).Treatment of peptide 2a with TCEP eliminates octamer formation and gives a downward streaking band at a position that corresponds to a tetramer (Fig. 5b).Disruption of the dimerization interface by N-  Covalent crosslinking of the antiparallel b-sheet dimer subunit observed in the X-ray crystallographic structure of peptide 2a stabilizes tetramer formation.Peptide 2d is a homologue of peptide 2b in which Val 36 is replaced with cysteine and crosslinked intermolecularly.Peptide 2d runs as a tetramer in SDS-PAGE (Fig. 5b).Upon treatment with TCEP, peptide 2d runs as bands that correspond to monomer and tetramer (Fig. 5b).The formation of the monomer upon reduction further correlates the tetramer assembly observed in SDS-PAGE with that observed in the crystal structure.The CD spectrum of peptide 2d resembles a b-sheet, suggesting that unlike peptide 2b, it folds as designed to adopt a b-hairpin like conformations (ESI Fig. S2 †).
We performed dynamic light scattering (DLS) studies to further characterize the oligomers formed by peptides 1a, 2a, and 2b in an aqueous environment without SDS.Under the same pH and buffer conditions of the CD experiments, DLS shows that these peptides form aggregates with hydrodynamic diameters of about 2 mm (ESI Fig. S4 †).The aggregates observed by DLS are far larger than the tetramer or octamer observed by SDS-PAGE, or the crystallographic tetramer formed by peptide 2a.These observations suggest that peptides derived from the bhairpin reported by Tycko and coworkers can assemble to form large assemblies in aqueous environments.

Cytotoxicity and membrane disruption of peptides 1a and 2a
1][42][43][44] Because the crystallographic b-barrel-like tetramer formed by peptide 2a resembles a pore, we speculated that peptides 1a and 2a may damage cell membranes through a pore-like mechanism of action.To investigate the cytotoxic and membrane disrupting potential of Ab derived peptides stabilized in the alignment reported by Tycko and coworkers, we carried out a caspase-3/7 activation assay and a dye-leakage assay using peptides 1a and 2a.Peptides 1a and 2a were thus found to activate caspase 3/7, an apoptotic marker, at 12.5-25 mM in SH-SY5Y cells (ESI Fig. S5 †).The dye-leakage assay was performed using negatively charged large unilamellar vesicles (LUVs) and peptides 1a and  The rest of the system is omitted for clarity.The simulation was set up using CHARMM-GUI, 54,55 and run for 500 ns at constant temperature and pressure using NMAD 2.14 (ref.56) with the CHARMM36 forcefield. 57,58Molecular graphics and simulation analyses were generated with VMD 1.9.3. 592a (ESI Fig. S6 †).In these experiments, peptides 1a and 2a disrupt anionic lipid membranes at 0.6-2.7 mM.Together, these data suggest that peptides 1a and 2a have limited potential to mimic some of the apoptotic and membrane-damaging behaviors of oligomers formed by full-length Ab.

Molecular dynamics simulation of an Ab tetramer in a lipid bilayer membrane
6][47][48][49] We thus generated a model of an Ab 9-42 bbarrel-like tetrameric assembly embedded in a fully hydrated POPC lipid bilayer from the crystallographic structure of peptide 2a and the REMD simulation and performed a 500 ns all-atom MD simulation (Fig. 6 and ESI S7 †).The incorporation of the tetramer in the lipid bilayer results in a reduction of the lipid bilayer thickness around the b-barrel-like assembly (ESI Fig. S8 †) as lipid phosphate groups and water molecules interact with polar moieties in the b-hairpins connecting loops and termini (Fig. 6 and ESI S9 †).Water molecules cross the membrane through the tetramer pore, and also interact with polar moieties on the outer surface of the tetramer (Fig. 6 and ESI S10 and S11 †).Although these results indicate that it is possible for a tetrameric assembly of peptide 2a to disrupt the membrane by forming pores, they cannot establish whether the caspase 3/7 activation and dye leakage observed for peptides 1a and 2a are caused by pore formation. 50,51The caspase 3/7, dyeleakage, and simulation data suggest that peptides 1a and 2a, and other peptides stabilized in the b-hairpin alignment described by Tycko and coworkers can interact with lipid membranes in a manner that our laboratory is beginning to explore. 52,53nclusion b-Hairpins are a key structural component of Ab oligomers and may contribute directly to oligomer heterogeneity through variation in hairpin alignment and topology.In this investigation, we studied the assembly and structure of oligomers formed by peptides derived from Ab 12-40 that were stabilized in the b-hairpin alignment reported by Tycko and coworkers.SDS-PAGE and X-ray crystallography reveal that Ab derived b-hairpins in this alignment assemble to form b-barrel-like tetramers that concatenate to form octamers.We envision that Ab bhairpins in other alignments might form other oligomers, with different structures, stoichiometries, and stabilities-and that all of these b-hairpins and oligomers exist in equilibrium.Earlier investigations from our laboratory on related Ab derived peptide systems have revealed the assembly of dimers, trimers, tetramers, hexamers, octamers, and dodecamers that differ substantially in assembly and structure.Further, mutations in familial Alzheimer's disease that alter the biophysical properties of the Ab peptide may have a signicant impact on Ab folding and assembly. 33,60These ndings would suggest that not all b-hairpins formed by the Ab peptide have an equal propensity to fold and assemble.
It is noteworthy that peptide 2a assembles to form oligomers of the same stoichiometry as the tetramer and octamer reported by Carulla and coworkers, but with signicant differences in structure. 4The tetramer reported by Carulla and coworkers is planar and does not form a b-barrel-like assembly; further the octamer is proposed to adopt a b-sandwich conformation.Neither the planar tetramer nor the b-sandwich contain a pore.Instead, the b-barrel-like tetramer formed by peptide 2a more closely resembles the hexameric "cylindrin" formed by peptide fragments of aB crystallin, reported by Eisenberg and coworkers. 613][64] The tetramers and octamers described herein provide additional models of how Ab may assemble into oligomers in membrane-like environments and the crystal state.DE-AC02-76SF00515.The authors thank Dr Robert Tycko for providing the coordinates of his Ab b-hairpin model (Fig. 1a).M. W. acknowledges the support from the Ministry of Science and Higher Education, Republic of Poland (Mobility Plus grant no.1647/MOB/V/2017/0).T. D. S. acknowledges the support from the University of California, Irvine, for funding through the Graduate Dean's Dissertation Year Fellowship, and thanks Alberto Smith and Victoria Sahrai for their assistance with peptide synthesis.

Fig. 1
Fig. 1 (a) Structures of Ab b-hairpins reported by Carulla and coworkers (PDB 6RHY), H ärd and Hoyer and coworkers (PDB 2OTK), and Tycko and coworkers (not deposited).(b and c) Chemical structures and cartoons of b-hairpin peptides derived from Ab 12-40 .

Fig. 2
Fig. 2 (a) Silver stained SDS-PAGE of peptides 1a-h, 2a-b, and Ab 40 and Ab 42 .SDS-PAGE was performed in Tris buffer at pH 6.8 with 2% (w/v) SDS on a 16% polyacrylamide gel with 50 mM solutions of peptide in each lane.(b and c) Circular dichroism (CD) spectra of peptides 1a-h and 2ab.CD spectra were acquired for each peptide at 50 mM in 10 mM phosphate buffer at pH 7.4; ellipticity was normalized for the number of residues in each peptide.Additional CD spectra for peptides 1i, 2c, and 2d are provided in the (ESI Fig. S2 †).

Fig. 3 X
Fig. 3 X-ray crystallographic structure of the b-barrel-like tetramer formed by peptide 2a (PDB 7RTZ).(a) Side view of the tetramer.(b) Top view of the tetramer.(c) Cartoon illustration of the parallel and antiparallel b-sheet interactions that stabilize the tetramer.(d) REMD simulation of a tetramer of Ab 9-42 based on the b-barrel-like tetramer formed by peptide 2a.Residues 12-22 and 30-40 are constrained to the crystallographic coordinates of peptide 2a.
methylation of Met 35 , in peptides 1i and 2c, also disrupts octamer formation (Fig. 5a and b) while partially or fully retaining propensities to adopt b-hairpin-like conformations (ESI Fig. S2 †).These experiments demonstrate that disruption of the hydrogen-bonding interfaces or destabilization of component b-hairpins disrupts octamer or tetramer formation.

Fig. 4 X
Fig. 4 X-ray crystallographic structure of the octamer formed by peptide 2a (PDB 7RTZ).(a) Two concatenated b-barrel-like tetramers form an octamer.(b) Assembly of the tetramers within the crystal lattice of peptide 2a to form concatenated chains of b-barrels.

Fig. 5
Fig. 5 (a) Silver stained SDS-PAGE of peptides 1a, 1b, 1i, and 1a after treatment with TCEP.(b) stained SDS-PAGE of peptides 2a-d, and 2a and 2d after treatment with TCEP.SDS-PAGE was performed in Tris buffer at pH 6.8 with 2% (w/v) SDS on a 16% polyacrylamide gel with 50 mM solutions of peptide in each lane.Treatment with TCEP was performed at 10 mM.

Fig. 6
Fig. 6 Configuration snapshots from an all-atom molecular dynamics simulation of a b-barrel-like tetramer formed by Ab 9-42 in a POPC lipid bilayer at 0 ns, 250 ns, and 500 ns.The Ab 9-42 tetramer is shown as a secondary structure representation.The lipid phosphate P atoms are shown as orange spheres, and the water molecules are shown in ball-andstick representations colored by atom (O, red; H, white).The rest of the system is omitted for clarity.The simulation was set up using CHARMM-GUI,54,55 and run for 500 ns at constant temperature and pressure using NMAD 2.14 (ref.56) with the CHARMM36 forcefield.57,58Molecular graphics and simulation analyses were generated with VMD 1.9.3.59