A尾(1-42) tetramer and octamer structures reveal edge conductivity pores as a mechanism for membrane damage AbstractFormation of amyloid-beta (A尾) oligomer pores in the membrane of neurons has been proposed to explain neurotoxicity in Alzheimer始s disease (AD). Here, we present the three-dimensional structure of an A尾 oligomer formed in a membrane mimicking environment, namely an A尾(1-42) tetramer, which comprises a six stranded 尾-sheet core. The two faces of the 尾-sheet core are hydrophobic and surrounded by the membrane-mimicking environment while the edges are hydrophilic and solvent-exposed. By increasing the concentration of A尾(1-42) in the sample, A尾(1-42) octamers are also formed, made by two A尾(1-42) tetramers facing each other forming a 尾-sandwich structure. Notably, A尾(1-42) tetramers and octamers inserted into lipid bilayers as well-defined pores. To establish oligomer structure-membrane activity relationships, molecular dynamics simulations were carried out. These studies revealed a mechanism of membrane disruption in which water permeation occurred through lipid-stabilized pores mediated by the hydrophilic residues located on the core 尾-sheets edges of the oligomers. IntroductionSubstantial genetic evidence links the amyloid-尾 peptide (A尾) to Alzheimer鈥檚 disease (AD)1. However, there is great controversy in establishing the exact A尾 form responsible for neurotoxicity. A尾 is obtained from a membrane protein, the amyloid precursor protein (APP), through the sequential cleavage of 尾- and 纬-secretase2. Upon APP cleavage, it is generally considered that A尾 is released to the extracellular environment. Due to its hydrophobic nature, A尾 then aggregates into multiple species, commonly referred to as soluble A尾 oligomers, which eventually evolve into A尾 fibrils3,4,5,6, the main component of amyloid plaques. Moreover, there is a less described pathway that considers that upon APP cleavage, a fraction of A尾 remains in the membrane evolving into membrane-associated A尾 oligomers, which would be directly responsible for compromising neuronal membrane integrity7.Since the amounts of A尾 fibrillar plaques do not correlate with cognitive decline8 researchers have focused on the study of both soluble and membrane-associated A尾 oligomers to identify the A尾 form responsible for neurotoxicity. Soluble A尾 oligomers have been prepared incubating synthetic A尾 samples under specific conditions hypothesized to stabilize a given A尾 oligomer form (culture media, pH, T, salts, 鈥? or engineering A尾 variants to lock the peptide in a conformation that is incompatible with fibril formation9. Many types of A尾 oligomers, such as ADDLs, amylospheroids, paranuclei or hexameric A尾42cc9 have been prepared using these approaches. In a similar manner, research dedicated to study A尾 in a membrane environment has used either detergent micelles10,11,12 or liposomes13,14,15,16. The study of A尾 in the presence of liposomes has been imaged by atomic force microscopy (AFM) revealing that A尾 incorporates into liposomes as oligomeric pores of different sizes15. Moreover, functional characterization of these samples using electrophysiological recordings in lipid bilayers demonstrated the presence of multiple single-channel currents of various sizes13,14,15. These results led to the proposal of the amyloid pore hypothesis nearly three decades ago13.However, in spite of the many efforts in this area, none of these studies have provided atomic structures for any A尾 oligomer. Without this information, it has not been possible to unequivocally establish A尾 oligomers鈥?mechanism of neurotoxicity or to design therapeutic strategies against their neurotoxic effects17. In 2016, we reported conditions to prepare homogenous and stable A尾 oligomers in membrane-mimicking environments18. We found that their formation was specific for A尾(1-42)鈥攖he A尾 variant most strongly linked to AD鈥? that they adopted a specific 尾-sheet structure, which is preserved in a lipid environment provided by bicelles, and that they incorporated into membranes exhibiting various types of pore-like behavior. Because of these properties, we named them 尾-sheet pore-forming A尾(1-42) oligomers (尾PFOsA尾(1-42)). Here we present the atomic structures of 尾PFOsA尾(1-42) by nuclear magnetic resonance (NMR) and mass-spectrometry (MS) and provide a mechanism for membrane disruption based on electrophysiology and simulation studies in membranes.Results尾PFOsA尾(1-42) sample comprise A尾(1-42) tetramersIn an earlier study, when developing conditions to prepare 尾PFOsA尾(1-42), we aimed at characterizing biologically relevant A尾 oligomers so we established conditions for their formation while working at pH 7.418,19. However, we also found that the oligomers adopted the same structure while being more stable when prepared at pH 9.0 (Supplementary Fig.聽1). Since structural characterization of 尾PFOsA尾(1-42) was facilitated when working with stable samples, we decided to work at pH 9.0. We prepared a selectively labeled 2H,15N,13C 尾PFOA尾(1-42) sample in dodecylphosphocholine (DPC) micelles at pH 9.0 and used high field NMR triple-resonance TROSY-type experiments to obtain sequence-specific resonance assignments (Supplementary Figs.聽2 and聽3). Peak assignment allowed us to establish that A尾(1-42) residues were observed in duplicate in the 2D [1H,15N]-TROSY spectrum (Fig.聽1a), which suggested that the sample comprised two distinct A尾(1-42) subunits. To highlight the detection of two A尾(1-42) subunits in the sample, residues belonging to each of them were identified as either red or green. Next, we used the C伪 and C尾 chemical shifts to derive the three-residue averaged (螖C伪-螖C尾) secondary chemical shifts to thus determine the presence of secondary structure elements in each A尾(1-42) subunit (Fig.聽1b). This analysis revealed that the red A尾(1-42) subunit contributed two 尾-strands, 尾1 and 尾2, to the oligomer structure. These strands extended, respectively, from G9 to A21 and from G29 to V40. Instead, in the green A尾(1-42) subunit, residues L17 to F20 exhibited 伪-helical propensity, while residues G29 to I41 adopted a 尾-strand conformation, referred to as 伪1 and 尾3, respectively. To finalize assignments, the connectivity between 尾1 and 尾2, and 伪1 and 尾3 secondary structural elements was established using mixtures of A尾(1-42) and A尾(17-42) with distinct isotope labels (Supplementary Fig.聽4).Fig. 1: Architecture of the A尾(1-42) tetramer.a Amide resonance assignments of the A尾(1-42) tetramer. Two A尾(1-42) subunits are detected and residues belonging to each of them are labeled in either red or green. b Three-bond averaged secondary chemical shifts versus residue number for the red (top) and the green (bottom) A尾(1-42) subunits. Secondary structural elements derived from chemical shift indices are shown at the top with its corresponding number. Arrows indicate 尾-strands and helical symbols helices. c Strips from a 3D NH-NH NOESY spectrum defining long-range intra-monomer interactions between the red A尾(1-42) subunit, long-range inter-monomer interactions between the red and the green A尾(1-42) subunits, and long-range inter-dimer interactions between the two green A尾(1-42) subunits. d The amino acid sequence of the A尾(1-42) tetramer is arranged on the basis of the secondary and tertiary structure. Amino acids in square denote 尾-sheet secondary structure as identified by secondary chemical shifts; all other amino acids are in circles. Blue lines denote experimentally observed NOE contacts between two amide protons. Bold lines indicate strong NOEs typically observed between hydrogen-bonded residues in 尾-sheets. Dashed lines show probable contacts between protons with degenerate 1H chemical shifts. The side chains of white and gray residues point towards distinct sides of the 尾-sheet plane, respectively. Orange circles correspond to residues that could not be assigned. Sample conditions were 1鈥塵M 2H,15N,13C A尾(1-42) in 10鈥塵M Tris, 28.5鈥塵M DPC at pH 9.0 after incubation for 24鈥塰 at 37鈥壜癈. Source data are provided as a Source data file.Full size imageNext, we used nuclear Overhauser effect spectroscopy (NOESY) to obtain long-range structural information. From the cross-peaks observed in the 3D NH-NH NOESY experiment, we identified eight NOEs between 尾1 and 尾2 strands of the red A尾(1-42) subunit and 7 NOEs between 尾2 strand of the red A尾(1-42) subunit and the 尾3 strand of the green A尾(1-42) subunit (Fig.聽1c). The observation of intra- and inter-subunit NOEs allowed us to establish the topology of an asymmetric dimer unit and to confirm that all the peaks detected in the 2D [1H,15N]-TROSY spectrum belonged to the same oligomer. Moreover, we also detected three NOEs involving residues of the 尾3 strand (Fig.聽1c), which could be explained only if two asymmetric dimer units interacted through 尾3 to form a tetramer in an antiparallel manner. All together, these NOEs allowed us to establish the complete topology of a six-stranded A尾(1-42) tetramer unit (Fig.聽1d). Moreover, since we did not detect any NOEs for the amide protons of 尾1 residues pointing outward of the 尾-sheet core (i.e., Y10, V12, H14, K16, V18, and F20), we inferred that the signals detected by NMR corresponded to an A尾(1-42) tetramer. To further validate the tetramer topology, we prepared specifically isotope-labeled samples and assigned the methyl groups of Ala, Ile, Leu, and Val (AILV) residues (Supplementary Fig.聽5). We then acquired 3D NH-CH3 NOESY and 3D CH3-CH3 NOESY spectra and obtained a network of 87 NH-CH3 and 25 CH3-CH3 NOEs consistent with the topology of the tetrameric unit (Supplementary Fig.聽6).NMR NOESY-type experiments allowed us to identify a network of more than 150 NOE contacts (Supplementary Table聽1) which, together with backbone dihedral angle (Supplementary Fig.聽7) and hydrogen-bond restraints, allowed us to define the structure of an A尾(1-42) tetramer (Fig.聽2a, Supplementary Fig.聽8a, b and Supplementary Table聽2). The tetramer comprised a 尾-sheet core made of six 尾-strands, connected by only two 尾-turns, leaving two short and two long, flexible N-termini, the latter comprising 伪1. The root mean square deviation (RMSD) of the A尾(1-42) tetramer ensemble was 0.77 and 1.34鈥壝?for the backbone and the heavy atoms of the six-stranded 尾-sheet core, respectively. Notably, all residues on both faces of the 尾-sheet core were hydrophobic except for three basic residues (i.e., H13, H14, and K16) located in 尾1, at the edges of the 尾-sheet core (Fig.聽2b). On the other hand, residues making the 尾-turns and the ends of the flexible N-termini were hydrophilic except for those comprising 伪1.Fig. 2: 3D structure of the A尾(1-42) tetramer prepared in DPC.a Ribbon diagram of the A尾(1-42) tetramer structure. A尾(1-42) subunits are colored either red or green to identify the asymmetric dimer unit that constitutes the building block of the A尾(1-42) tetramer. b Distribution of hydrophobic and charged residues on the surface of the A尾(1-42) tetramer. Hydrophobic residues are white, polar are yellow, and positively and negatively charged are red and blue, respectively. c Water accessibility of amide protons revealed through 2D [1H,15N]-HSQC spectra obtained at different pHs and through measurement of amide temperature coefficients. Solvent accessibility is linearly coded on the basis of the intensity of blue, with light blue corresponding to low water accessibility and dark blue corresponding to high water accessibility. Unassigned residues are shown in gray. d DPC accessibility of amide protons. The residues that showed NOEs between the backbone amide proton and the N-bound methyls of the choline head group of DPC are shown in green. The amide residues that showed paramagnetic enhancement, 蔚, upon addition of 16-DSA are shown in magenta. The 蔚 values are linearly coded on the basis of the intensity of magenta, with light pink corresponding to 蔚鈥?鈥? and dark magenta corresponding to 蔚鈥?鈥壩?sub>max. e Solvent Accessible Surface Area (SASA, 脜2) from MD simulations of the A尾(1-42) tetramer in DPC. Detergent micelle is represented as a smoothed transparent surface. The figure was prepared with the program Pymol. Source data are provided as a Source data file.Full size imageA尾(1-42) tetramer鈥擠PC interactionHaving established the 3D structure of the A尾(1-42) tetramer, we examined how it interacted with the surrounding media, namely water and the DPC detergent molecules. 2D [1H,15N]-HSQC spectra were acquired at pH 8.5 and 9.5 (Supplementary Fig.聽9a, b). Residues belonging to the 尾-sheet core and some belonging to 伪1 were detected at both pHs, while some of the 伪1 residues and those corresponding to the 尾-turns and the N-termini ends were detected only when the spectrum was measured at the lowest pH. This observation suggested that residues comprising the 尾-turns and the N-termini ends exchanged faster with the solvent and were therefore more exposed than those making the 尾-sheet core and 伪1 (Fig.聽2c). To establish whether the more protected 尾-sheet core residues exhibited distinct degrees of solvent protection, we determined their amide temperature coefficients (螖未/螖T). Most of the NH amide protons of residues comprising 尾1, 尾2, and 尾3 were the most affected by temperature changes, which is consistent with these residues forming stable hydrogen bonds20. In contrast, amide protons of 尾1 residues pointing out of the 尾-sheet core (i.e., Y10, V12, H14, K16, V18, and F20) exhibited the lowest amide temperature coefficients, suggesting that these residues are the most water accessible of all residues comprising the 尾-sheet core (Fig.聽2c).Next, to characterize the interaction of the DPC molecules with the surface of the A尾(1-42) tetramer, we acquired a 3D 15N-resolved [1H,1H]-NOESY spectrum of the A尾(1-42) tetramer using a selectively 13C methyl-protonated AILV and otherwise uniformly 2H,15N A尾(1-42) sample prepared using DPC at natural isotopic abundance. Analysis of this spectrum allowed us to identify two types of intermolecular interactions. First, we detected intermolecular NOEs between residues V12, L17, and L18, located in 尾1, and the N-bound methyl groups of the choline head group of DPC (Fig.聽2d and Supplementary Fig.聽10). Notably, this observation suggested that the detergent head groups bent towards the positively charged side chain of K16 located at the hydrophilic edges of the 尾-sheet core in order to stabilize them. Second, we detected intermolecular NOEs between all amide protons comprising the 尾-sheet core and the hydrophobic tail of DPC, with the largest intensities for residues located at the center of the 尾-sheet core and decreasing toward its edges (Fig.聽2d and Supplementary Fig.聽10). These observations were confirmed using a paramagnetic labeled detergent, 16-doxyl stearic acid (16-DSA) (Fig.聽2d and Supplementary Figs.聽11鈥?a data-track=\"click\" data-track-label=\"link\" data-track-action=\"supplementary material anchor\" href=\"/articles/s41467-020-16566-1#MOESM1\">13).Finally, the interaction of the A尾(1-42) tetramer with DPC micelles was further studied through molecular simulations using the SimShape approach21. Over the course of a 1鈥塶s nonequilibrium simulation, the A尾(1-42) tetramer was enveloped in a toroidal DPC micelle (Supplementary Fig.聽14). Afterwards, the toroidal complex was equilibrated in explicit solvent for 60鈥塶s. During this time, the hydrophobic terminal tail carbon of DPC was observed to interact predominantly with the two faces of the six-stranded 尾-sheet core, while transient contacts were also detected with the 伪1 region. Additionally, the DPC polar head was observed to interact with the hydrophilic edges of the six-stranded 尾-sheet region, which slowly became exposed to the solvent (Fig.聽2e). Finally, these interactions were further validated by simulating the equilibrated protein-detergent complex in the absence of any external biasing forces (Supplementary Fig.聽15). In summary, the experimental and the simulation results indicate that both faces of the central hydrophobic 尾-sheet core of the A尾(1-42) tetramer were covered with a monolayer of DPC with 伪1 residues also interacting with the hydrophobic tail of DPC. In contrast, the rest of the residues, including the hydrophilic edges of the 尾-sheet core, were solvent-exposed and further stabilized by interactions with the polar head of DPC.尾PFOsA尾(1-42) sample contain A尾(1-42) tetramers and octamersPrevious electrical recordings using planar lipid bilayers had revealed that the 尾PFOsA尾(1-42) sample induced various types of pore-like behavior18. Having established the 3D structure of the A尾(1-42) tetramer, it was difficult to envision how it could be directly responsible for pore formation. For this reason, we attempted to determine whether other oligomer stoichiometries, not detectable by NMR, were present in the 尾PFOsA尾(1-42) sample. To this end, we set to analyze the sample by means of size exclusion chromatography coupled to native ion mobility mass spectrometry (SEC/IM-MS)22. This strategy presented a unique opportunity to establish the stoichiometry of the potentially distinct oligomer species present as a function of their elution through a SEC column. We had previously analyzed 尾PFOsA尾(1-42) in a SEC column equilibrated in DPC and shown that the sample eluted as a major peak at 27.4鈥塵L (Fig.聽3a). However, to carry out SEC/IM-MS, a different detergent that would be compatible with MS analysis and would preserve oligomer stability was required23. C8E5 was found to fulfill both requirements (Fig.聽4a). MS analysis of the early eluting volume of the 尾PFOsA尾(1-42) peak revealed charge states consistent with the presence of tetramers and octamers. Analysis of the late eluting volume showed an increase in the relative abundance of the charge states corresponding to tetramers relative to those assigned to octamers. Importantly, the use of IM prior to MS analysis allowed unambiguous assignment of the contribution of distinct oligomer stoichiometries to each charge state (Supplementary Fig.聽16 and Supplementary Table聽3). This analysis led us to conclude that, although in agreement with NMR experiments, the stoichiometry of the major species present in the 尾PFOsA尾(1-42) sample was A尾(1-42) tetramers; A尾(1-42) octamers were also present. In addition, since no charge states specific for other oligomer stoichiometries between tetramers and octamers were detected, these results suggested that tetramers were the building block for octamer formation. Notably, upon increasing activation conditions of the mass spectrometer, octamers did not decrease significantly in the spectrum at the maximum activation conditions afforded by the instrument (Supplementary Fig.聽17), indicating that octamers were not derived from the forced co-habitation of two tetramers in a micelle but rather from specific interactions between the A尾 subunits composing it.Fig. 3: 尾PFOA尾(1-42) samples can be enriched in either tetramers or octamers.SEC of 尾PFOsA尾(1-42) prepared at low (a) and high (d) A尾(1-42) concentration in a column equilibrated in DPC. The peaks labeled in orange and blue are assigned, respectively, to A尾(1-42) tetramers and octamers. SDS-PAGE analysis of 尾PFOsA尾(1-42) prepared at low (b) and high (e) A尾(1-42) concentrations either not cross-linked (ctrl) or having been previously cross-linked (XL) and showing the effect of boiling (+) and non-boiling (鈭?. MALDI-TOF analysis of 尾PFOsA尾(1-42) prepared at low (c) and high (f) A尾(1-42) concentrations. Data shown in panels b and e are representative from three independent experiments. Source data are provided as a Source data file.Full size imageFig. 4: A尾(1-42) octamers adopt a 尾-sandwich structure.SEC-MS analysis of 尾PFOsA尾(1-42) prepared at low (a) and high (b) A尾(1-42) concentration. To couple SEC to MS analysis, the SEC column was equilibrated in C8E5. The mass spectra extracted from the blue and orange SEC peaks are shown, respectively, with a blue and orange line on top of them. The charge states corresponding to monomers, dimers, trimers, tetramers, and octamers are indicated with schematic drawings and labeled, respectively, in black, pink, yellow, orange and blue. c A尾(1-42) tetramer structure derived from NMR restraints before (solid orange line) and after gas phase simulation for 100鈥塶s prior to CCS calculation (dashed orange line). d 尾-sandwich octamer model based on the interaction of two A尾(1-42) tetramers before (solid blue line) after gas phase simulation for 100鈥塶s prior to CCS calculation (dashed blue line). e Experimental CCS of the tetramer (black dots) compared to the theoretical CCS of the A尾(1-42) tetramer structure before (solid orange line) and after gas phase simulation for 100鈥塶s prior to CCS calculation (dashed orange line). f Experimental CCS of the octamer (black dots) compared to the theoretical CCS of the 尾-sandwich structure before (solid blue line) and after gas phase simulation for 100鈥塶s prior to CCS calculation (dashed blue line). Source data are provided as a Source data file.Full size imagePreparation of 尾PFOsA尾(1-42) enriched in A尾(1-42) octamersHaving detected A尾(1-42) octamers in the 尾PFOsA尾(1-42) sample, we attempted to enrich our sample in this oligomer form to pursue its characterization. To this end, we maintained the concentration of DPC micelles constant and increased the concentration of A尾(1-42) to mimic the consequences of an increase of the latter in the membrane7. Thus, from this point, we worked with two 尾PFOsA尾(1-42) samples, one corresponding to the sample analyzed up to now and prepared at 150鈥壩糓 of A尾(1-42), referred to as 尾PFOsLOW_A尾(1-42), and one prepared at 450鈥壩糓 A尾(1-42), referred to as 尾PFOsHIGH_A尾(1-42). To establish whether 尾PFOsHIGH_A尾(1-42) were enriched in octameric forms, we analyzed them by SEC using a column equilibrated in DPC (Fig.聽3d). This analysis resulted in a major peak eluting 1.4鈥塵L earlier than 尾PFOsLOW_A尾(1-42), as well as a small peak eluting at the same volume as the major peak detected for 尾PFOsLOW_A尾(1-42). These findings indicated that working at high A尾(1-42) concentration indeed led to the formation of a larger oligomer.To study the stoichiometry of the oligomers present in the two samples, after preparing them in DPC micelles without any buffer exchange, we submitted them to chemical crosslinking. Given the abundance of basic and acid moieties in the flexible regions of the A尾(1-42) tetramer structure derived by NMR (Supplementary Fig.聽8c), we decided to generate zero-length (ZL) cross-links between Lys and Asp or Glu residues using DMTMM as a coupling reagent24. As previously described, SDS-PAGE analysis of the non-cross-linked 尾PFOsLOW_A尾(1-42) sample led, depending on whether the sample had been previously boiled or not, to either a 5鈥塳Da band, corresponding to A尾(1-42) monomers, or to a major band at 18鈥塳Da, consistent with A尾(1-42) tetramers (Fig.聽3b)18. In contrast, SDS-PAGE analysis of the cross-linked 尾PFOsLOW_A尾(1-42) sample led to a major band at 14鈥塳Da, regardless of whether it had been boiled previously. The decrease in migration detected for the cross-linked samples is associated with protein compaction caused by crosslinking events25. To further confirm the stoichiometry of the cross-linked bands established by SDS-PAGE, samples were analyzed by MALDI-TOF (Fig.聽3c). MALDI ionization involves harsh conditions, which prevents preservation of the non-covalent interactions present in protein complexes. Therefore, as expected, the molecular weight of the sample analyzed by MALDI-TOF without being cross-linked led to the detection of a peak corresponding to the molecular mass of the monomer (Supplementary Table聽4). Instead, analysis of the cross-linked 尾PFOsLOW_A尾(1-42) sample led to the detection of a major peak consistent with the mass of an A尾(1-42) tetramer, thereby confirming the suitability of the ZL chemistry to efficiently cross-link the major species formed under this condition. Next, we applied the same cross-linking chemistry to the analysis of the 尾PFOsHIGH_A尾(1-42) sample. SDS-PAGE analysis of the non-cross-linked samples led to the same bands obtained for 尾PFOsLOW_A尾(1-42), as well as to a faint band at about 30鈥塳Da, consistent with A尾(1-42) octamers (Fig.聽3e). Instead, SDS-PAGE analysis of the cross-linked 尾PFOsHIGH_A尾(1-42) sample, both non-boiled and boiled, led to the detection of bands migrating at 28鈥塳Da, consistent with A尾(1-42) octamer formation. This result was further validated by MALDI-TOF analysis (Fig.聽3f). All together, these results indicated that the 尾PFOsHIGH_A尾(1-42) sample comprises mainly A尾(1-42) octamers. Moreover, the observation that SDS-PAGE analysis of the non-cross-linked and non-boiled 尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42) samples led to mainly the same A尾(1-42) tetramer band points to A尾(1-42) octamers being formed by two tetrameric building blocks whose stabilizing interactions are not preserved in the presence of SDS.A尾(1-42) octamers adopt a 尾-sandwich structureSubsequently, we analyzed 尾PFOsHIGH_A尾(1-42) by SEC/IM-MS. Although C8E5, the detergent required for native MS analysis, did not completely stabilize the larger oligomer detected in a SEC column equilibrated in DPC (compare Figs.聽3d to 4b), analysis of the early eluting peak, corresponding to the larger oligomeric species, led almost exclusively to three charge states assigned to A尾(1-42) octamers (Fig.聽4b, Supplementary Figs.聽18 and 19, and Supplementary Table聽3). In summary, characterization of the 尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42) samples by SEC, cross-linking/MALDI-TOF and SEC/IM-MS revealed that the former was enriched in A尾(1-42) tetramers and the latter in octamers.To study the conformational state of the A尾(1-42) octamers, we used IM-MS to derive their collision cross-sections (TWCCSN2) (Supplementary Fig.聽20 and Fig.聽4c鈥揻). We first validated this approach working with A尾(1-42) tetramers, for which we had determined their 3D structure by NMR. Since a certain degree of compaction is expected in the gas phase26, we simulated in vacuo the most representative charge state (+6) for the A尾(1-42) tetramer structure for 100鈥塶s. We observed a quasi-immediate and significant degree of compaction (gyration radius reduced by ~30%) that remained stable over the remaining 100鈥塶s (Supplementary Fig.聽21a). Compaction could be mainly attributed to the flexible N-termini ends while the 尾-sheet core remained stable throughout the simulation (Supplementary Fig.聽21b). Next, we compared the experimental TWCCSN2 for the A尾(1-42) tetramer (1598 脜2) with the theoretical CCS obtained for the A尾(1-42) tetramer structure determined by NMR after gas phase simulation (1647 脜2) (Fig.聽4c, e). This agreement between experimental and average CCS values after MD simulation validated the use of IM-MS to obtain insights into the structure of A尾(1-42) octamers.SDS-PAGE analysis of non-boiled samples enriched in A尾(1-42) octamers led to a major band at 18鈥塳Da as obtained for samples enriched in A尾(1-42) tetramers (Fig.聽3b, e). This result indicated that A尾(1-42) octamers are derived from the assembly of two A尾(1-42) tetramers. Consequently, to study the conformational state of the A尾(1-42) octamers, we considered octamer models built from the assembly of two A尾(1-42) tetramers. Considering the structure of the latter, we examined the association of two tetramers to either form a loose 尾-barrel or a 尾-sandwich structure. To this end, we simulated in vacuo for 100鈥塶s the most representative charge state (+8) of both A尾(1-42) octamer models. The behavior of the 尾-barrel and 尾-sandwich structure along the simulation was very different (Supplementary Fig.聽21). The 尾-sandwich structure showed a significant and quasi-immediate degree of compaction attributed to the flexible N-termini ends, as the 尾-sheet content remained stable to 40% throughout the simulation. Instead, the 尾-barrel structure compacted in three steps along the first 15鈥塶s as a result of not only the flexible N-termini but also an immediate destabilization of its core 尾-sheets as shown by the ~20% decrease in 尾-sheet content. Since the theoretical CCS of the 尾-sandwich octamer (2546 脜2) immediately matched the experimental TWCCSN2 for the A尾(1-42) octamer (2469 脜2) (Fig.聽4f) and its compacted structure remained stable in vacuo, as observed for the A尾(1-42) tetramers and as expected for membrane protein鈥搈icelle complexes in vacuo27, we considered the 尾-sandwich structure the relevant topology for the A尾(1-42) octamers. This result is indeed consistent with the physicochemical properties of the A尾(1-42) tetramer as its two hydrophobic faces do not support its self-assembly in a 尾-barrel octamer structure with a central hydrophilic cavity. Instead, the A尾(1-42) tetramer assembly in a 尾-sandwich octamer fully fulfils its physicochemical properties.Structures of 尾PFOsA尾(1-42) reveal edge conductivity poresHaving obtained the means to prepare and characterize 尾PFOsA尾(1-42) samples enriched in tetramers and octamers, we set to compare their activity in lipid bilayers by electrical recordings using planar lipid bilayers (Supplementary Fig.聽22). The only difference between the two samples was found in the occurrence rate of the different pore-like behaviors with 尾PFOsLOW_A尾(1-42), enriched in tetramers, exhibiting fast and noisy transitions with undefined open pore conductance values for a higher number of times than 尾PFOsHIGH_A尾(1-42), enriched in octamers, and the latter exhibiting a well-defined open pore with no current fluctuations for a higher number of times than the former. The observation of pore-like activity for A尾(1-42) tetramer and octamer samples motivated the use of molecular dynamics (MD) simulations to probe the mechanism of bilayer disruption at an atomistic scale. These simulations involved the application of an external electric field to observe ion conductance properties in 150鈥塵M NaCl, 310鈥塊, at 100鈥塵V for 500鈥塶s.We first monitored the conformational drift, structural flexibility, and secondary structural content of the A尾(1-42) tetramer and octamer structures along the simulation time. RMSD of the C伪 atoms of the tetramer and octamer structures revealed plateau levels of 2 and 2.5鈥壝? respectively (Supplementary Fig.聽23). Analysis of the root mean square fluctuation (RMSF) showed the greatest fluctuations for the N-termini of both the red and green subunits and the loop region connecting 尾1 and 尾2 strands of the red subunit while almost no fluctuations were detected for the 尾1, 尾2, and 尾3 strands (Supplementary Fig.聽24). Finally, analysis of 尾-sheet content of the membrane-bound A尾(1-42) tetramer and octamer revealed that it remained stable along the course of the simulation (Supplementary Fig.聽25). All together, these simulations and analyses indicated that the overall fold of the oligomers remained stable in a membrane bilayer environment with an applied electric field.Next, we aimed at gaining insights into the mechanism by which A尾(1-42) tetramer and octamer structures promoted bilayer disruption at an atomistic scale. The presence of hydrophilic residues on the edges of both the A尾(1-42) tetramer and octamer structures resulted in their unfavorable exposure to the hydrophobic lipid tails of the membrane (Fig.聽5). This situation led to lipid rearrangement, such that the head groups of the lipids reoriented to face the hydrophilic edges. Contacts between protein and DPPC head group atoms were characterized for both tetramer and octamer systems. This analysis revealed that the headgroup of DPPC assembled towards the hydrophilic edges, specifically 尾1 strands, of the tetramers and octamers (Supplementary Figs.聽26 and 27) leading to the formation of lipid-stabilized pores, which stabilized the protein鈥搇ipid complex. Subsequently, we analyzed water permeation profiles along the membrane normal (z) direction. We observed a higher degree of water permeation and a greater solvent-accessible surface area in the octamer than in the tetramer (Fig.聽5). We associate the formation of lipid-stabilized pores observed during the MD simulations with the mechanism of water and ion permeation observed experimentally through electrical recordings using planar lipid bilayers (Supplementary Fig.聽22) and propose them to explain the neurotoxicity observed in AD through the disruption of cellular ionic homeostasis.Fig. 5: MD simulations in DPPC membrane bilayer of A尾(1-42).a tetramer and b octamer. The snapshots (two top rows) and water permeation profiles represented as histograms of the distribution of water molecules along the membrane normal (z) direction (bottom row) correspond to the initial coordinates (left), after 100鈥塶s isothermal-isobaric NPT equilibrium simulation (middle), and after 500鈥塶s canonical ensemble NVT simulation with 100鈥塵V applied electric field (right). Protein is shown in grey, DPPC headgroup phosphorous atoms are shown in tan, and water in red/white. The tan lines in the water permeation profiles represent approximately the membrane spanning region.Full size imageDiscussionIn this study, we have used a multidisciplinary approach comprising the use of NMR, MS, electrophysiology, and MD simulations. This combined approach has allowed us to identify a putative A尾 form potentially responsible for AD neurotoxicity, as we have defined the structural and biophysical properties of membrane-associated A尾(1-42) tetramers and octamers. To date, only the 3D structures of A尾 fibrils have been described3,4,5,6 and no experimental structure has been reported for A尾 oligomers, only models. Compared to the structure of A尾 fibrils3,4,5,6, the oligomers characterized in this study offer a 3D arrangement for A尾(1-42) completely different: from the intermolecular formation of parallel 尾-sheets in A尾 fibrils to intramolecular and intermolecular antiparallel 尾-sheet formation in the membrane-associated A尾(1-42) oligomers.Compared to previously reported A尾 oligomer models, our NMR structure of A尾(1-42) tetramers exhibits some similarities but also some differences. Thus, as in our tetramer structure, previous models of A尾(1-42) peptides simulated in an implicit membrane28 or revealed by solid-state NMR spectroscopy of an engineered variant that forms stable protofibrils9 are based on a 尾-hairpin topology with residues D23 to K28 constituting the connecting loop. However, the topology of residues K16-A42, which in these models are organized in 尾-hairpins is different from the one presented in this study. While in our A尾(1-42) tetramer structure residues G10-A21 and G29-V40 form two 尾-strands, 尾1 and 尾2, in the above described previous models, residues K16-G36 adopt a 尾-hairpin conformation and residues G39-I41 at the C-terminus form a third shorter 尾-strand following a turn involving residues G37-G38. Moreover, in these previous studies a single A尾(1-42) conformation is considered in the simulations or is observed by solid-state NMR while in our study A尾(1-42) adopts two distinct conformations in the tetramer structure. Finally, while A尾(1-42) oligomers from solid-state NMR were modeled as circular hexamers9, simulations of 尾-hairpin oligomers showed that A尾(1-42) peptides could assemble as stable double-layered 尾-sheets with lateral association of 尾-hairpins in a parallel manner28,29. Similarly, our A尾(1-42) octamers associate in double-layered 尾-sheets but with antiparallel association of 尾-hairpins. Therefore, our study widens the description of the much-needed low energy structural landscape of A尾.In addition, a strong link of this study to AD comes from the fact that the formation of the tetramers and octamers reported here is specific for A尾(1-42), the variant most strongly linked to AD, versus A尾(1-40), the variant most abundantly produced18. Having obtained the 3D structure for A尾(1-42) tetramers allows to rationalize why its formation is specific for A尾(1-42). Indeed, the absence of the two C(t) residues shortens the 尾-strands comprising the six-stranded 尾-sheet core from 14 residues to 12, which is too short to span the hydrophobic portion of the lipid bilayer, thus preventing its stability in a membrane environment and providing a structural explanation to understand the different pathological role of A尾(1-42) and A尾(1-40) in AD.One important implication of this study is to establish whether the results obtained in a micelle environment can be extrapolated to the lipid bilayer. To address this important point we will consider three scenarios: (i) insertion of A尾(1-42) tetramers and octamers from a micelle to a lipid bilayer; (ii) formation of A尾(1-42) tetramers and octamers within the lipid bilayer; and, (iii) stability of A尾(1-42) tetramers and octamers in the lipid bilayer. The process of A尾(1-42) tetramers and octamers insertion from a micelle to a lipid bilayer occurs in our electrophysiology experiments (Supplementary Fig.聽22). The results from these experiments indicate that both samples enriched in A尾(1-42) tetramers and octamers lead to specific pore-like behavior. In these experiments, assuming the structure observed in the micelle is maintained in the lipid bilayer, the N-termini of the A尾(1-42) tetramers and octamers, with all their charged residues, are required to traverse through the hydrophobic core of the bilayer. This process, which is observed for the insertion of pre-assembled nanopores, such as ClyA and FraC nanopores into lipid bilayers, can be very efficient30. For example, entire proteins including GF, 尾-lactamase, organophosphorus hydrolase, and 尾-galactosidase can be fused to the N-terminus of a ClyA nanopore and transported across a lipid bilayer in vivo31. In the case of A尾(1-42) tetramers and octamers, the unstructured nature of the charged N-termini would help traversing the bilayers as demonstrated for cationic cell-penetrating peptides32. In a cellular environment, we cannot rule out the possibility that the insertion of such structures is facilitated by other cellular components. However, systems such as the Sec machinery that aid membrane insertion usually require proteins with a signal peptide and they operate one monomer at the time. Hence, such a mechanism would not progress through oligomeric intermediates. Our results indicate that A尾(1-42) tetramers and octamers can form pores in the hydrophobic environment of a lipid bilayer, but we cannot exclude that the complexity of biological membranes will affect the kinetics and thermodynamics of biological membrane insertion.As per the formation of A尾(1-42) tetramers and octamers in the native lipid bilayer, it is generally accepted that after APP cleavage A尾 is released from the membrane. However, several studies indicate that A尾 might accumulate in the membrane. Indeed, larger amounts of A尾 are detected in the membrane fraction than in the soluble one7,33. We therefore propose that A尾 accumulation in the membrane, as it occurs within micelles, could be the trigger for formation of A尾(1-42) tetramers and octamers in the native environment of lipid bilayers. Finally, evidence for the stability of 尾PFOsA尾(1-42) in a lipid environment come from a previous study where we showed structural stability in the lipid environment provided by bicelles18. Moreover, in this study we have performed long time-scale molecular dynamics (MD) simulations with applied electric field, which have revealed that both A尾(1-42) tetramers and octamers maintain their overall fold.Finally, apart from establishing the structure of membrane-associated A尾(1-42) tetramers and octamers and assessing their pore-activity in planar lipid bilayers, MD simulations revealed that membrane disruption arises from the hydrophilic residues located on the edges of the 尾-sheets leading to the formation of lipid-stabilized pores. Such behavior resembles the toroidal pore-type behavior shown by many antimicrobial peptides34 and would also be consistent with the reported antimicrobial activity for A尾35,36. Moreover, the role of hydrophilic residues in membrane disruption is consistent with previous studies that show small penalties to expose charged side chains, such as Lys and Arg, to lipids due to the stabilizing influence of membrane deformations for the protonated form37,38.In summary, we have established the 3D structure of an A尾 membrane-associated oligomer with the ability to form lipid-stabilized pores that could explain neurotoxicity in AD. We therefore present a unique opportunity to establish whether the A尾(1-42) tetramers and octamers described in this study are indeed the A尾 species responsible for AD neurotoxicity. For example, by producing antibodies that specifically recognize them and subsequently using these antibodies to validate the A尾(1-42) tetramer and octamer structures in AD brains. Therefore, the oligomers whose structure and function are described in this paper can be the long-sought A尾 species responsible for AD.MethodsReagentsLipids and detergents were purchased from Avanti Polar Lipids or Affymetrix-Anatrace. Deuterated reagents were purchased from Cortecnet or Eurisotop. All other reagents were supplied by Sigma-Aldrich unless otherwise stated. Kits for selective isotopically labeled samples were purchased from NMR-Bio. All buffers and solutions were freshly prepared using water provided by a Milli-Q system (18鈥塎W鈥塩m鈭? at 25鈥壜癈, Millipore).Purification of synthetic A尾 samples and A尾 mixturesA尾(1-42) and A尾(17-42) were synthesized and purified by Dr. James I. Elliott (New Haven, CT, USA). The following protocol, described for A尾(1-42) but applicable to the other A尾 peptides under study, was used to obtain A尾 in a monomeric state. In all, 5鈥?0鈥塵g of lyophilized synthetic A尾(1-42) peptide were resuspended in 6.8鈥塎 guanidinium thiocyanate (GdnSCN) to a final concentration of 2.5鈥塵g A尾(1-42)鈥塵L鈭? and sonicated for 5鈥塵in in an ice bath. Afterwards, the sample was further diluted with Milli-Q water to 1.5鈥塵g A尾(1-42)鈥塵L鈭? and 4鈥塎 GdnSCN, and centrifuged. Finally, 2鈥塵L of the 1.5鈥塵g A尾(1-42)鈥塵L鈭? solution was injected into a HiLoad Superdex 30 prep grade column (GE Healthcare), previously equilibrated with 50鈥塵M ammonium carbonate. The fractions corresponding to monomeric A尾(1-42) were collected and their purity and concentration were determined by Reversed Phase High Performance Liquid Chromatography (RP-HPLC). The pool was finally aliquoted in the desired amounts, freeze-dried, and kept at 鈭?0鈥壜癈 until use.For mixed samples containing different peptides such as A尾(1-42)/A尾(17-42), we purified, as described above, the most insoluble peptide first and prepared aliquots in the desired amounts, froze them with liquid nitrogen, and kept them at 鈭?0鈥壜癈. Afterwards the second peptide was purified in the same way, aliquots were added on top of the already frozen one and the combined aliquot was freeze-dried. Samples were kept at 鈭?0鈥壜癈 until use.Expression and purification of recombinant A尾 samplesThe DNA encoding A尾(1-42) was synthesized by PCR following KOD polymerase (Novagen) methods and using a modular approach39, but with the following primers to add the 15鈥塨p on each side for the In-Fusion method:Fw 5鈥?GCGAACAGATCGGTGGTGATGCGG-A-GTTCCGTCATGATTCAG-3鈥?andRev 5鈥?ATGGTCTAGAAAGCTTTATTACG-CTATGACAACACCACCCACCATGAG-TCCAATGATGGCACC-3鈥?/p>The amplified DNA fragment was purified and cloned into a pOPINS vector40 previously cut with KpnI and HindIII (New England Biolabs) restriction enzymes following the In-Fusion cloning method (Clontech). This resulted in a plasmid for the expression of A尾(1-42) in the cytoplasm of Rosetta (DE3) pLysS E. coli cells (Novagen) as a fusion protein with an N-terminal hexahistidine SUMO affinity tag.For all labeling schemes, Rosetta (DE3) pLysS E. coli cells (Novagen) were transformed with the expression vector and grown overnight at 37鈥壜癈 on Luria Bertani (LB)-agar plates containing 1% glucose. All cell cultures were also supplemented with 35鈥壩糶鈥塵L鈭? chloramphenicol and 50鈥壜礸鈥塵L鈭? kanamycin. To enhance protein production, all A尾 peptides were expressed with the construct (His)6-SUMO-A尾 using SUMO as a fusion partner. An auto-induction procedure was used to produce [U-15N] A尾(1-42) and [U-15N] A尾(17-42)41. Briefly, single colonies were picked and grown overnight in LB supplemented with 1% glucose. The pre-culture was centrifuged, and the pellet was transferred to 15N-labeled P-5052 auto-inducing media with the appropriate antibiotics. The resulting cultures were grown for 6鈥塰 at 37鈥壜癈. The temperature was then lowered to 25鈥壜癈, and the culture was incubated for a further 22鈥塰. The cells were then harvested by centrifugation and frozen at 鈭?0鈥壜癈.M9 minimal medium was used to produce [U-2H,13C,15N] A尾(1-42) and [U-2H,15N] A尾(1-42) following previously reported protocols41. Briefly, single colonies were picked and grown overnight in LB supplemented with 1% glucose. Cells containing the DNA construct were adapted to grow in minimal medium in a stepwise manner by inoculating the cells into fresh M9 minimal medium containing increasing percentages of D2O. The final pellet, already grown overnight in M9 minimal medium prepared using 100% D2O, was re-suspended and inoculated in 1鈥塋 M9 medium also prepared using 100% D2O and containing 1鈥塯鈥塋鈭? 15NH4Cl and 2鈥塯鈥塋鈭? d-glucose-13C6鈭?,2,3,4,5,6,6-d7 or d-glucose-1,2,3,4,5,6,6-d7. The culture was grown at 37鈥壜癈 and induced at an OD600 ~1 by the addition of IPTG to a final concentration of 0.5鈥塵M. After overnight growth at 25鈥壜癈, the cells were harvested by centrifugation and then frozen at 鈭?0鈥壜癈.For the production of selectively labeled Ile-[13CH3]未1, Ala-[13CH3], Leu/Val-[13CH3]proR A尾(1-42) samples, we followed previously published procedures42. Briefly, 2-[13CH3], 4-[2H3] acetolactate (NMR-Bio) at 300鈥塵g鈥塵L鈭? was added 1鈥塰 prior to induction. Forty minutes later (20鈥塵in prior to induction), 2-hydroxy-2-(1鈥?[2H2], 2鈥?[13C])ethyl-3-keto-4-[2H3]butanoic acid (NMR-Bio) at 60鈥塵g鈥塵L鈭? and 2-[2H], 3-[13C]alanine (NMR-Bio) at 700鈥塵g鈥塵L鈭? were added. Protein expression was induced with IPTG.The following protocol, described for A尾(1-42) but applicable to the other A尾 peptides under study, was used to purify and obtain A尾 in a monomeric state. After protein expression, cells were lysed by sonication and centrifuged, and the supernatant was then purified as already described41. Briefly, the cleared soluble fraction was loaded onto a HisTrap HP 5-mL Ni column (GE Healthcare) and the fusion protein was eluted with 0.5鈥塵M imidazole. IMAC fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and those containing the fusion protein were pooled. Next, the buffer was exchanged using a HiPrep 26/10 desalting column (GE Healthcare) equilibrated with 50鈥塵M ammonium carbonate and 1鈥塵M TCEP. Afterwards, the concentration and purity of protein was determined by Nanodrop庐 and RP-HPLC. Subsequently, samples were incubated overnight at 4鈥壜癈 with SUMO protease (Ulp1) in a 1:50 protease:protein ratio to cleave A尾(1-42) from the SUMO fusion tag. The concentration of A尾(1-42) peptide after the cleavage was determined by RP-HPLC analysis. Subsequently, aliquots containing 3.75鈥塵g A尾(1-42) were prepared and freeze-dried. Each of these aliquots was solubilized with 6.8鈥塎 GdnSCN to 2.5鈥塵g A尾(1-42)鈥塵L鈭? and sonicated for 5鈥塵in in an ice bath. Afterwards, the sample was further diluted with Milli-Q water to 1.5鈥塵g A尾(1-42)鈥塵L鈭? and 4鈥塎 GdnSCN, and then centrifuged. Finally, 2.5鈥塵L of the 1.5鈥塵g A尾(1-42) mL鈭? solution was injected into a HiLoad Superdex 30 prep grade column (GE Healthcare), previously equilibrated with 50鈥塵M ammonium carbonate. The peaks corresponding to SUMO and monomeric A尾(1-42) were collected separately, and their purity and concentration were determined by RP-HPLC. The pool containing pure A尾(1-42) was aliquoted in the desired amounts, freeze-dried, and kept at 鈭?0鈥壜癈 until use.Isotope-labeled samples for NMR experimentsThe following samples were produced: [U-15N]-A尾(1-42), [U-15N]-A尾(17-42) for 2D 1H-15N-HSQC experiments; [U-2H,13C,15N]-A尾(1-42) for 3D backbone assignment experiments; [U-2H,13C,15N]-Ile-[13CH3]未1, Ala-[13CH3], Leu/Val-[13CH3]proR-A尾(1-42) for side chain methyl assignments; and [U-2H,15N]-Ile-[13CH3] 未1, Ala-[13CH3], Leu/Val-[13CH3]proR-A尾(1-42) for 3D 13CH3-13CH3 and NH-13CH3 NOESY experiments.Preparation of 尾PFOsA尾(1-42) sample for NMR experimentsFor NMR experiments, 尾PFOsA尾(1-42) were prepared by dissolving lyophilized isotopically labeled monomeric A尾(1-42) in the required volume of 10鈥塵M Tris-d11 and 28.5鈥塵M DPC-d38 to reach 1鈥塵M A尾(1-42). Afterwards, the pH was checked and adjusted to pH 9.5 or 8.5 with either a 10% HCl or a 10% NaOH solution, and the sample was left incubating at 37鈥壜癈 for 24鈥塰. To prepare 尾PFOsA尾42 at other A尾(1-42) concentrations, only the concentration of DPC micelles ([MDPC]) was adjusted so that the final [A尾(1-42]/[MDPC] ratio was 2:1, where [MDPC] is the concentration of DPC micelles and equals to the difference between the DPC detergent concentration ([DDPC]) and its critical micellar concentration (CMC) divided by its aggregation number (i.e. ([DDPC]-CMC)/aggregation number)). The CMC of DPC was taken to be 1.5鈥塵M10,43 and the DPC aggregation number 5443. This preparation, which is later referred to in the paper as 尾PFOsLOW_A尾(1-42), was found to be enriched mainly in A尾(1-42) tetramers.NMR experimentsAll experiments were carried out at 37鈥壜癈 on a 900鈥塎Hz Bruker Avance III HD spectrometer equipped with a 5-mm CP-TCI cryogenic probe, or an 800鈥塎Hz Bruker Avance III HD spectrometer equipped with a 3-mm CP-TCI cryogenic probe, both instruments located at the Swedish NMR Centre in Gothenburg, or an 800鈥塎Hz Bruker Avance III HD spectrometer equipped with a 5-mm CP-TCI cryogenic probe, located at IECB in Bordeaux.For the resonance assignment of backbone atoms of the A尾(1-42) tetramer prepared in DPC micelles, experiments from the standard Bruker library were recorded (HNCA, HNCACB, HNCO, and HN-NH NOESY). For the resonance assignment of methyl groups, experiments from the standard Bruker library were recorded (Hme)Cme([C]CA)CO, (Hme)Cme([C]CA)NH, Hme(Cme[C]CA)NH44 and complemented with (H)C-TOCSY-C-TOCSY-(C)H experiments45. Additionally, four 3D SOFAST-NOESY-HMQC experiments44 were recorded to obtain NOE correlation between methyl groups (Hm-HmCm and Cm-HmCm) and between methyl and amide protons (Hm-NH and Cm-HN) of the A尾(1-42) tetramer in DPC micelles. The acquisition parameters for all the NMR experiments carried out are summarized in Supplementary Table聽5. All these experiments were acquired using non-uniform sampling (NUS) using TopSpin 3.5, processed with MDDNMR 2.5 software45,46, and analyzed using CCPNmr Analysis 2.4.2.NMR amide temperature coefficientsAmide temperature coefficients of the A尾(1-42) tetramer were determined by measuring the 2D [1H,15N]-TROSY spectra of the A尾(1-42) tetramer sample at 303, 310, 317, and 324鈥塊 on a 900鈥塎Hz Bruker Avance III HD spectrometer equipped with a 5-mm CP-TCI cryogenic probe, and calculated using Eq. (1):$${mathrm{Temperature}};{mathrm{coefficient}};{mathrm{values}} = Delta {updelta}_{{mathrm{NH}}}/Delta {mathrm{T}}$$ It is well established that, in aqueous solvents, exposed NHs typically display gradients from 鈭? to 鈭?.5鈥塸pb K147 while hydrogen-bonded exchange-protected NHs are characterized less negative 螖未/螖T values than 鈭?鈥塸pb K1. However, numerous exceptions to these generalizations occur. In our case, the first anomalous observation was the fact that all amide protons presented positive 螖未/螖T, meaning that chemical shifts of amide proton resonances shift downfield as the temperature increases. These downfield shifts with increased temperature may be explained by greater solvent protection of NH protons47, probably due to the effect of the detergent micelle surrounding the tetramer. Furthermore, we observed that most of the NH amide protons of residues from 尾1, 尾2, and 尾3 were the most affected by temperature changes. Cierpicki et al. noted that amides involved in hydrogen bonds with a length of less than ~3.0鈥壝?exhibited a larger temperature coefficient, because the secondary chemical shift caused by hydrogen bonding is greater, and so the same fractional change gives rise to a larger gradient20. This report would be consistent with residues with the largest 螖未/螖T being involved in stable hydrogen bonds.NMR titrations聽with paramagnetic reagentsNMR titrations with 16-DOXYL-stearic acid (16-DSA) were performed by addition of concentrated stock solutions of 16-DSA to an A尾(1-42) tetramer NMR sample. Stock solutions of 16-DSA were obtained by dissolving this chemical in methanol-d4. The A尾(1-42) tetramer NMR sample was prepared at 1鈥塵M A尾(1-42), using the appropriately labeled sample, in 10鈥塵M Tris-d11, 28.5鈥塵M DPC-d38 at pH 9.5. 16-DSA stock solution was subsequently added to this A尾(1-42) sample to obtain the following concentrations of 16-DSA: 0.3, 0.45, 0.6, 1.2, and 2.4鈥塵M. [1H,15N]-TROSY and [1H,13C]-HMQC experiments were acquired at each 16-DSA concentration point. These NMR experiments were also performed on a sample without 16-DSA in order to be used as reference.NMR structure calculation of the A尾(1-42) tetramerThe structure of the A尾(1-42) tetramer was determined with the iterative ARIA 2.3.2/CNS 1.21 software48,49. Distance restraints were derived from NOE cross-peaks (HN-HN, HN-Methyl, Methyl-Methyl) and used as input for ARIA 2.3.2, together with dihedral angle restraints and hydrogen-bond restraints. Upper bound distances for NOE restraints were derived from NOE cross-peaks volumes using characteristic distances (sequential NH-NH NOEs in 尾-sheet or intra-residual NH-Methyl in Alanines). Backbone dihedral angles were predicted from backbone chemical shifts with TALOS-N 4.2150. Predictions classified as strong were converted to dihedral angle restraints with an error corresponding to twice the standard deviation given by TALOS-N 4.21. Hydrogen bond restraints for anti-parallel beta-strand pairing were deduced from the NOE pattern and confirmed by initial calculations from NOEs and dihedral angle restraints only. Each hydrogen bond is encoded by two restraints (HN鈥 with upper-bound 2.3鈥壝?and N鈥, upper-bound 3.3鈥壝?. During structure calculation, four copies of an A尾(1-42) chain were modeled, using NCS restraints to maintain each dimer superimposable in the tetramer. For each iteration, 100 conformations were generated, except for the last iteration, where 500 conformers were calculated. The 50 lowest-energy conformers were refined in a shell of DMSO molecules51 and the 15 refined conformers with the least number of distance restraint violations were selected as the final A尾(1-42) tetramer ensemble. The structure ensemble was validated with PROCHECK 3.5.452 WHATIF 8.353 and MolProbity 3.1954. The coordinates for the A尾(1-42) tetramer structure have been deposited in the Protein Data Bank with accession code 6RHY.Simulations of A尾(1-42) tetramer solubilization in DPCFrom the NMR ensemble of A尾(1-42) tetramer structures determined with ARIA, the conformer with the least restraint violations was selected to be solubilized in n-dodecylphosphocholine (DPC) micelle. The SimShape protocol with NAMD 2.13 was used to accelerate the assembly of the protein鈥搈icelle complex21. This method uses grid-steered molecular dynamics to assemble detergents into a toroidal micelle that wraps around the hydrophobic core of a membrane protein. Three biasing potentials were utilized in total. Two took the shape of concentric toroids, where the first toroid was coupled to the head-group heavy atoms of DPC, while the second smaller toroid was coupled to the tail-heavy atoms. The third biasing potential took the shape of a plane and was coupled to the detergent tail-heavy atoms.Prior knowledge of the approximate micelle shape and orientation facilitated the design of the shape of the toroidal potentials. HDX studies characterized the central six-stranded 尾-sheet region of the tetramer with slow water exchange, suggesting burial of these residues within the micelle. This ~29鈥壝?long region of the A尾(1-42) tetramer was used to inform the dimensions of the toroid-shaped grid potentials. The number of DPC molecules in the micelle was determined to be 120 by estimating the size of the tetramer-micelle complex using overall correlation time obtained from NMR18. The DPC molecule positions were initialized in a toroidal pattern around the protein such that there was a minimum distance of 10鈥壝?between any given pair of detergent and protein atoms.The complex was simulated using NAMD with grid-steered molecular dynamics for 1鈥塶s at 310鈥塊 with a Langevin thermostat with a damping coefficient of 5鈥塸s. Generalized Born Implicit Solvent (GBIS) with ionic strength 0.15鈥塵M and dielectric constant of 80 were used. CHARMM36 forcefield parameters were used. Head and tail atoms were separately coupled to their grid potentials with scaling factors of 0.18 and 0.25, respectively. The tail atoms were additionally coupled to the planar grid potential with a scaling factor of 0.14. Backbone heavy atoms were harmonically restrained using a 1鈥塳cal (mol鈥壝?sup>2)鈭? spring constant to maintain protein structure during detergent assembly. After 1鈥塶s, detergent molecules assembled into a micelle around the protein with a toroidal shape similar to the attractive grid potentials (Supplementary Fig.聽14).Continuing with the SimShape protocol, the micellar complex was completely uncoupled from the toroidal attractive potentials, solvated with TIP3P water, and ionized with 150鈥塵M NaCl. The solvated system was minimized with conjugate gradient energy minimization for 5000 steps, and simulated for 30鈥塶s with protein backbone heavy atom harmonic position restraints with a spring constant of 1鈥塳cal鈥?mol鈥壝?sup>2)鈭?. Next, the protein restraints were gradually removed to allow for complete equilibration of the complex (Fig.聽2e). The spring constant was lowered in steps by 0.001鈥塳cal (mol鈥壝?sup>2)鈭? every 10鈥塸s for 10鈥塶s. Finally, eight replicates of the unrestrained protein鈥搈icelle complex were simulated for an additional 100鈥塶s each with no biasing forces applied. These equilibrium trajectories were then used to characterize contacts between the tetramer and detergents.Contacts were calculated between the A尾(1-42) tetramer and DPC molecules and are shown in Supplementary Fig.聽15. Two contact sites on DPC were considered: the head group nitrogen atom and the terminal tail carbon atom. The number of contacts was defined as the number of contact sites within 9鈥壝?of an amide backbone nitrogen atom and was calculated for every frame, summed over symmetric chains, and averaged over a 100-ns trajectory, returning an average number of contacts per residue. The per-residue average contact number of the eight independent replicates was then considered to be eight independent samples, allowing for calculation of statistical error per residue, shown as 卤standard deviation divided by the number of independent samples.Preparation of 尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42)尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42) corresponding to A尾(1-42]/[MDPC] ratios of 2:1 and 6:1, respectively, were prepared from freeze-dried monomeric A尾(1-42) samples and dissolved in 10鈥塵M Tris, 5.5鈥塵M DPC adjusted to pH 9, reaching a final concentration of 150鈥壩糓 A尾(1-42) in the case of 尾PFOsLOW_A尾(1-42) and 450鈥壩糓 A尾(1-42) in the case of 尾PFOsHIGH_A尾(1-42). The samples were incubated at 37鈥壜癈 for 24鈥塰.SEC尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42) samples were injected into a tandem Superdex 200 increase 10/300 (GE Healthcare). The columns were equilibrated with 10鈥塵M Tris, 100鈥塵M NaCl at pH 9 containing 3鈥塵M DPC and eluted at 4鈥壜癈 at a flow rate of 0.5鈥塵L鈥塵in鈭?. Data was collected and analyzed using Unicorn v7.0 (GE Healthcare).SEC/IM-MSAn Acquity UPLC H-class system (Waters, Manchester, UK) comprising a quaternary solvent manager, a sample manager set at 10鈥壜癈, a column oven and a TUV detector operating at 280鈥塶m and 214鈥塶m was coupled to a Synapt G2 HDMS mass spectrometer (Waters) for online SEC/IM-MS instrumentation22. An Acquity BEH SEC column (4.6鈥壝椻€?50鈥塵m, 1.7 渭m particle size, 200鈥壝?pore size) (Waters) was equilibrated with 200鈥塵M (NH4)2CO3, 14.2鈥塵M C8E5 at pH 9.0 and run with the following flow rate gradient: 0.25鈥塵L min鈭? over 4鈥塵in; then 0.10鈥塵L min鈭? over 6鈥塵in and finally 0.25鈥塵L min鈭? over 2鈥塵in.The IM-MS experiments are reported as recommended by Gabelica et al55. The Synapt G2 HDMS was operated in positive mode with a capillary voltage of 3.0鈥塳V. The main parameters鈥攕ample cone 180鈥塚, trap collision energy 100鈥塚, trap gas flow 5鈥塵L鈥塵in鈭? and backing pressure 6鈥塵bar鈥攚ere finely tuned to disrupt the detergent protein interaction and to maintain the oligomer species. Acquisitions were performed in the m/z range 1000鈥?0,000 with a 1.5-s scan time. External calibration was performed using singly charged ions produced by a 2鈥塯鈥塋鈭? solution of cesium iodide dissolved in 2-propanol/water (50/50, v/v). To assess the effect of activation energy on the oligomeric species detected, we increased the energy conditions by means of the sampling cone (100, 180 and 200鈥塚) and the trap collision energy (50, 100 and 160鈥塚). MS data collection and analysis were performed using MassLynx 4.1 (Waters).In order to unambiguously assign the oligomeric state of each MS peak and to measure the drift time of each species, an ion mobility method using travelling wave IMS (TWIMS) technology was optimized as described below. Prior to TWIMS separation, ions were thermalized in the helium cell (180鈥塵L鈥塵in鈭?). Subsequently, ion separation was performed in the pressurized ion mobility cell using a constant N2 (purity鈥?gt;鈥?9%) flow rate of 90鈥塵L鈥塵in鈭?. The wave height and velocity were 40鈥塚 and 800鈥塵鈥塻鈭?, respectively. Transfer collision energy was set to 15鈥塚 to extract the ions from the IM cell to the TOF analyzer. IM-MS experiments were performed in triplicate under identical instrumental conditions. Ion mobility data was collected and analysed using DriftScope 2.4 (Waters). We assessed the CCS from the mobility measurements using Eq. (2)56:$${mathrm{CCS}} = frac{3}{{16}}sqrt {frac{{2{uppi}}}{{{upmu k}}_{mathrm{B}}{mathrm{T}}}} frac{{{mathrm{ze}}}}{{{mathrm{NK}}}} = frac{3}{{16}}sqrt {frac{{2{uppi}}}{{{upmu k}}_{mathrm{B}}{mathrm{T}}}} frac{{{mathrm{ze}}}}{{{mathrm{N}}_0left( {frac{{mathrm{p}}}{{{mathrm{p}}_0}}frac{{{mathrm{T}}_0}}{{mathrm{T}}}} right){mathrm{K}}}} = frac{3}{{16}}sqrt {frac{{2{uppi}}}{{{upmu k}}_{mathrm{B}}{mathrm{T}}}} frac{{{mathrm{ze}}}}{{{mathrm{N}}_0{mathrm{K}}_0}}$$ IM data were calibrated to perform CCS calculations using the most intense charge state of external calibrants prepared under non-denaturing conditions. The choice of calibrants is a critical point in the calibration framework57. The calibrants used were cytochrome C, 尾-lactoglobulin monomer and dimer, and avidin, which were in the same range in terms of MW, z, and CCS as those of the A尾(1-42) tetramers and octamers used in this study (Supplementary Fig.聽20). Theoretical CCS for our samples were derived from atomic coordinates of the NMR structure of the A尾(1-42) tetramer and two octamer models built using the structure of the A尾(1-42) tetramer as a building block. The first octamer model was based on the association of two tetramers to form a loose 尾-barrel structure and the second one on the association of two tetramers in a 尾-sandwich structure. Theoretical CCS value for the A尾(1-42) tetramer and octamers were calculated using the previously described structures after 100鈥塶s MD simulation in the gas phase (Supplementary Fig.聽21). Gas phase simulations were prepared using the default vacuum simulation parameters in the QwikMD plugin of VMD 1.9.4a37, and extended to 100鈥塶s. The QwikMD prepared gas phase simulations were ran using NAMD 2.13, with the CHARMM36 forcefield, using a 2.0 femtosecond time step, and at a temperature of 300鈥塊. The two most abundant charges states for the tetramer (+6) and octamer (+8) were chosen as ionization states. The results depicted in Fig.聽4 were obtained with a charge distribution considering that the oligomers were prepared at pH 9.0 and that during positive electrospray only residues not protected by the micelle got protonated. To establish that charge location was not biasing the experiments two additional charge distributions consistent with the tetramer (+6) and octamer (+8) were built and simulated in the gas phase for tetramer and octamers, for a total of 100鈥塶s of gas phase simulations. Snapshots of the simulation trajectories were taken every 2鈥塸s, and used to obtain theoretical CCS values using the Projection Approximation method within the Impact 1.0 software58. Final values for all three charge distributions were within 60鈥壝?sup>2 of their mean values for CCS calculations of tetramers and octamers. Their corresponding PDB files were used as an input file and were run on Impact with a converGEnce value of 1% enabled to determine an average CCS as a mean of three independent calculations. Solvent and heteroatoms were excluded for the theoretical calculation.Cross-linking of 尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42)尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42) were prepared as described in the section Preparation of 尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42), with the exception that 10鈥塵M sodium carbonate (Na2CO3) was used instead of 10鈥塵M Tris to avoid the interference of Tris in the cross-linking reaction. After sample preparation, the concentration of 尾PFOsLOW_A尾(1-42) was maintained at 150鈥壩糓 A尾(1-42) while that of 尾PFOsHIGH_A尾(1-42) was brought from 450鈥壩糓 to 150鈥壩糓 A尾(1-42) by diluting it with a solution containing 10鈥塵M Na2CO3, 1.5鈥塵M DPC at pH 9. Afterwards, both samples were cross-linked using (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)鈭?-methyl-morpholinium chloride) (DMTMM) as the cross-linking reagent24. DMTMM was added to a final concentration of 15鈥塵M (4.15鈥塵g鈥塵L鈭?) and the samples were incubated for 2鈥塰 at 50鈥壜癈 and 800鈥塺pm. Samples were quenched by directly preparing them for SDS-PAGE and high-mass MALDI analysis.SDS-PAGE analysis of cross-linked oligomer samplesThe cross-linked samples were diluted to 50鈥壜礛 A尾(1-42) using a solution of 10鈥塵M Na2CO3 and 1.5鈥塵M DPC at pH 9. Finally, 20鈥壜礚 of the resulting solution was mixed with 10鈥壜礚 of 3X sample buffer (3X SB) and 20鈥壜礚 of the mixture, either non-boiled or boiled (for 5鈥塵in at 95鈥壜癈) were electrophoresed in 1-mm thick SDS-PAGE gels containing 15% acrylamide. Gels were run at 50鈥塚 for 30鈥塵in, 120鈥塚 for 2鈥塰 and stained using Coomassie Blue.High-mass MALDI-MS analysis of cross-linked oligomer samplesBefore MALDI-TOF analysis, cross-linked samples were diluted down to 37.5鈥壩糓 A尾(1-42) in H2O. This dilution step is critical to reduce the amount of detergent that could later interfere with the co-crystallization of the sample with the matrix, which is an essential prerequisite for the MALDI ionization process59. Next, diluted samples were mixed (1:1鈥塿/v) with a matrix solution of sinapic acid (10鈥塵g鈥塵L鈭?) containing (1:1鈥塿/v) acetonitrile/deionized water with 0.1% trifluoroacetic acid (TFA). Each mixture (2鈥壜祃 thereof) was deposited on the MALDI target plate using the dried-droplet method. As a control, 2鈥壜祃 of 尾PFOsLOW_A尾(1-42) and 尾PFOsHIGH_A尾(1-42) samples prepared as described but without adding the cross-linking reagent were examined using the same deposition method. High-mass MALDI-MS analyses were carried out on a MALDI-TOF mass spectrometer (Autoflex III, Bruker) used in linear mode and equipped with a HM3 high-mass detector (CovalX AG), which allows the (sub-碌M) detection of macromolecules up to 1500鈥塳Da with low saturation. Calibration was achieved using singly and doubly charged bovine serum albumin ions ([M鈥?鈥?鈥塇]2+= 33216鈥塂a and [M鈥?鈥塇]+鈥?鈥?6431鈥塂a) and the gas phase dimer of this protein ([2鈥塎鈥?鈥塇]+鈥?鈥?32861鈥塂a). The mass spectra were acquired by averaging 2000 shots (8 different positions into each spot and 250 shots per position), using the same laser fluency before and after crosslinking. The spectra were processed (including background subtraction and smoothing) using FlexAnalysis 3.4.Electrical recordings with planar lipid bilayersIonic currents from planar bilayers formed from diphytanoyl-sn-glycero-3-phosphocholine in 10鈥塵M Tris路HCl and 150鈥塵M NaCl at pH 7.5 and 23鈥壜癈 were measured by applying a 2鈥塳Hz low-pass Bassel filter with a 10鈥塳Hz sampling rate. Potentials were applied, and the current was recorded using Ag/AgCl electrodes connected to a patch-clamp amplifier (Axopatch 200B, Axon Instruments). Current recordings were analyzed using the Clampfit 10 software package (Molecular devices). Open-pore currents were measured by a Gaussian fit to all-point histogram. The center of the peak corresponds to the open-pore conductance and the width at half height to the error. Each electrophysiology chamber contained 500鈥壩糒 10鈥塵M Tris路HCl and 150鈥塵M NaCl at pH 7.5. Two samples were analyzed, 尾PFOsLOW_A尾(1-42), which was diluted from 1:250 to 1:100 in the chamber18, and 尾PFOsHIGH_A尾(1-42), which was diluted 1:100 in the chamber. For 尾PFOsLOW_A尾(1-42), type 1, 2, and 3 pores were observed in 17%, 48%, and 35% of the experiments (N鈥?鈥?05). For 尾PFOsHIGH_A尾(1-42), type 1, 2, and 3 pores were observed in 8.5%, 35%, and 56% of the experiments (N鈥?鈥?1). Controls were carried out to establish that the concentration of the detergent micelles present in the samples did not affect the stability of the bilayer.A尾(1-42) tetramer and octamer simulations in a DPPC bilayerMolecular dynamics simulations of the A尾(1-42) tetramer and octamer in planar lipid bilayers were performed under an applied electric field using NAMD 2.13. The protonation state of the titrable amino acids was chosen as that expected at pH 7.4 (lysine and arginine positively charged and aspartic and glutamic negatively charged). The ionization state of all histidine sidechains were set to neutral, using the HSD parameters of the CHARMM36m forcefield. Two structures were simulated, each in triplicate, one corresponding to the A尾(1-42) tetramer structure obtained by NMR and the other to the octamer 尾-sandwich structure determined by CCS. These structures were aligned to the principal axes of their 尾-sheet regions, and embedded into 80脜x80脜 planar 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers using the CHARMM-GUI input generator and CHARMM36m force field60. The systems were further simulated at equilibrium NPT conditions for 100鈥塶s with semi-isotropic pressure coupling in the X鈥揧 plane. The systems were then equilibrated in an NVT ensemble for 10鈥塶s. Then an external electric field of 100鈥塵V was applied along the Z-axis and each system was simulated for another 500鈥塶s.Secondary structure content for the membrane-bound A尾(1-42) tetramer and octamer was also calculated for the simulations with applied electric field. For each system, the dictionary of protein secondary structure (DSSP) assignments were calculated at each frame using the MDtraj 1.9.3 python package61. The helix content was defined as the fraction of residues in 伪-helix conformation, while the 尾-sheet content was defined as the fraction of residues in a 尾-strand conformation. The secondary structure content for each replicate of the tetramer and octamer systems was obtained. For each system, the average 尾-sheet content of each replicate simulation was calculated, allowing for calculation of statistical error, which is shown as 卤standard deviation between the three replicates (Supplementary Fig.聽25).Water permeation profiles were also calculated at three timepoints along the membrane-bound simulations: the initial (post-minimization, pre-equilibration) structure, after 100鈥塶s equilibrium simulation, and after 500鈥塶s simulation with applied electric field. These histograms represent the distribution of water along the membrane normal (z) direction. These water permeation profiles were then averaged over the three replicates of each system, and the average shown in Fig.聽5.Contacts between the bilayer DPPC molecules and the A尾(1-42) tetramer and octamer during the applied 100鈥塵V electric field were calculated. Two contact groups on DPPC were considered: the headgroup nitrogen and the two terminal tail carbon atoms. The number of contacts was defined as the number of contact sites within 9鈥壝?of an amide backbone nitrogen atom and was calculated for every frame, summed over symmetric chains, and averaged over the last 100鈥塶s of the 500鈥塶s trajectory (Supplementary Figs.聽26 and 27), returning an average number of contacts per residue. The per-residue average contact number of the three independent replicates was then considered three independent samples, allowing for calculation of statistical error per residue, shown as 卤standard deviation divided by the number of independent samples.Reporting summaryFurther information on research design is available in the聽Nature Research Reporting Summary linked to this article. Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. A reporting summary for this Article is available as a聽Supplementary Information file. The source data underlying Figs.聽1a, b, 2c, d, 3a鈥揻, 4a, b, e, f and Supplementary Tables S3 and S4 are provided as a Source data file. Code availability Accession codes for deposited data: coordinates have been deposited in the Protein Data Bank under accession number PDB 6RHY, and chemical shifts have been deposited in the Biological Magnetic Resonance Bank under entry 34396. References1.Selkoe, D. J. Hardy, J. The amyloid hypothesis of Alzheimer鈥檚 disease at 25 years. EMBO Mol. Med. 8, 595鈥?08 (2016).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 2.Haass, C., Kaether, C., Thinakaran, G. Sisodia, S. Trafficking and proteolytic processing of APP. Cold Spring Harb. Perspect. Med. 2, a006270鈥揳006270 (2012).PubMed聽 PubMed Central聽 Article聽 CAS聽Google Scholar聽 3.Bai, X.-C. et al. An atomic structure of human 纬-secretase. Nature 525, 212鈥?17 (2015).ADS聽 CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 4.W盲lti, M. A. et al. Atomic-resolution structure of a disease-relevant A尾(1鈥?2) amyloid fibril. Proc. Natl Acad. Sci. USA 113, E4976鈥揈4984 (2016).PubMed聽 Article聽 CAS聽Google Scholar聽 5.Colvin, M. T. et al. Atomic resolution structure of monomorphic A尾42 amyloid fibrils. J. Am. Chem. Soc. 138, 9663鈥?674 (2016).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 6.Gremer, L. et al. Fibril structure of amyloid-尾(1-42) by cryo-electron microscopy. Science 358, 116鈥?19 (2017).ADS聽 CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 7.Roberts, B. R. et al. Biochemically-defined pools of amyloid-尾 in sporadic Alzheimer鈥檚 disease: correlation with amyloid PET. Brain 140, 1486鈥?498 (2017).PubMed聽 Article聽Google Scholar聽 8.Dickson, D. W. et al. Correlations of synaptic and pathological markers with cognition of the elderly. Neurobiol. Aging 16, 285鈥?98 (1995). Discussion 298鈥?04.CAS聽 PubMed聽 Article聽Google Scholar聽 9.Lendel, C. et al. A Hexameric peptide barrel as building block of amyloid-尾 Protofibrils. Angew. Chem. Int. Ed. 53, 12756鈥?2760 (2014).CAS聽 Article聽Google Scholar聽 10.Mandal, P. K. Pettegrew, J. W. Alzheimer鈥檚 disease: soluble oligomeric A尾(1鈥?0) peptide in membrane mimic environment from solution NMR and circular dichroism studies. Neurochem Res. 29, 1鈥? (2004).Article聽Google Scholar聽 11.Tew, D. J. et al. Stabilization of neurotoxic soluble beta-sheet-rich conformations of the Alzheimer鈥檚 disease amyloid-beta peptide. Biophys. J. 94, 2752鈥?766 (2008).ADS聽 CAS聽 PubMed聽 Article聽Google Scholar聽 12.Yu, L. et al. Structural characterization of a soluble amyloid 尾-peptide oligomer. Biochemistry 48, 1870鈥?877 (2009).CAS聽 PubMed聽 Article聽Google Scholar聽 13.Arispe, N., Rojas, E. Pollard, H. B. Alzheimer disease amyloid beta protein forms calcium channels in bilayer membranes: blockade by tromethamine and aluminum. Proc. Natl Acad. Sci. USA 90, 567鈥?71 (1993).ADS聽 CAS聽 PubMed聽 Article聽Google Scholar聽 14.Hirakura, Y., Lin, M. C. Kagan, B. L. Alzheimer amyloid a尾1鈥?2 channels: Effects of solvent, pH, and congo red. J. Neurosci. Res. 57, 458鈥?66 (1999).CAS聽 PubMed聽 Article聽Google Scholar聽 15.Lin, H., Bhatia, R. Lal, R. Amyloid beta protein forms ion channels: implications for Alzheimer鈥檚 disease pathophysiology. FASEB J. 15, 2433鈥?444 (2001).CAS聽 PubMed聽 Article聽Google Scholar聽 16.Butterfield, S. M. Lashuel, H. A. Amyloidogenic protein-membrane interactions: mechanistic insight from model systems. Angew. Chem. Int. Ed. 49, 5628鈥?654 (2010).CAS聽 Article聽Google Scholar聽 17.Benilova, I., Karran, E. De Strooper, B. The toxic A尾 oligomer and Alzheimer鈥檚 disease: an emperor in need of clothes. Nat. Neurosci. 15, 349鈥?57 (2012).CAS聽 PubMed聽 Article聽Google Scholar聽 18.Serra-Batiste, M. et al. A尾42 assembles into specific 尾-barrel pore-forming oligomers in membrane-mimicking environments. Proc. Natl Acad. Sci. USA 113, 10866鈥?0871 (2016).CAS聽 PubMed聽 Article聽Google Scholar聽 19.Serra-Batiste, M. et al. Preparation of a well-defined and stable 尾-barrel pore-forming A尾42 oligomer. Methods Mol. Biol. 1779, 13鈥?2 (2018).CAS聽 PubMed聽 Article聽Google Scholar聽 20.Cierpicki, T. Otlewski, J. Amide proton temperature coefficients as hydrogen bond indicators in proteins. J. Biomol. NMR 21, 249鈥?61 (2001).CAS聽 PubMed聽 Article聽Google Scholar聽 21.Wang, Y, Tajkhorshid, E. The Simshape Method for Protein-Detergent Interaction Research.聽Biophys. J. 114, 679A (2018).22.Ehkirch, A. et al. Hyphenation of size exclusion chromatography to native ion mobility mass spectrometry for the analytical characterization of therapeutic antibodies and related products. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 1086, 176鈥?83 (2018).CAS聽 Article聽Google Scholar聽 23.Laganowsky, A., Reading, E., Hopper, J. T. S. Robinson, C. V. Mass spectrometry of intact membrane protein complexes. Nat. Protoc. 8, 639鈥?51 (2013).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 24.Leitner, A. et al. Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes. Proc. Natl Acad. Sci. USA 111, 9455鈥?460 (2014).ADS聽 CAS聽 PubMed聽 Article聽Google Scholar聽 25.Sitkiewicz, E., Ol臋dzki, J., Pozna艅ski, J. Dadlez, M. Di-Tyrosine cross-link decreases the collisional cross-section of A尾 peptide dimers and trimers in the gas phase: an ion mobility study. PLoS ONE 9, e100200鈥揺100214 (2014).ADS聽 PubMed聽 PubMed Central聽 Article聽 CAS聽Google Scholar聽 26.Pagel, K., Natan, E., Hall, Z., Fersht, A. R. Robinson, C. V. Intrinsically disordered p53 and its complexes populate compact conformations in the gas phase. Angew. Chem. Int. Ed. Engl. 52, 361鈥?65 (2013).CAS聽 PubMed聽 Article聽Google Scholar聽 27.Friemann, R., Larsson, D. S. D., Wang, Y. van der Spoel, D. Molecular dynamics simulations of a membrane protein-micelle complex in vacuo. J. Am. Chem. Soc. 131, 16606鈥?6607 (2009).CAS聽 PubMed聽 Article聽Google Scholar聽 28.Strodel, B., Lee, J. W. L., Whittleston, C. S. Wales, D. J. Transmembrane structures for Alzheimer鈥檚 A尾(1-42) oligomers. J. Am. Chem. Soc. 132, 13300鈥?3312 (2010).CAS聽 PubMed聽 Article聽Google Scholar聽 29.Poojari, C., Kukol, A. Strodel, B. How the amyloid-尾 peptide and membranes affect each other: an extensive simulation study. Biochim. Biophys. Acta 1828, 327鈥?39 (2013).CAS聽 PubMed聽 Article聽Google Scholar聽 30.Maglia, G., Heron, A. J., Stoddart, D., Japrung, D. Bayley, H. Analysis of single nucleic acid molecules with protein nanopores. Methods Enzymol. 475, 591鈥?23 (2010).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 31.Kim, J.-Y. et al. Engineered bacterial outer membrane vesicles with enhanced functionality. J. Mol. Biol. 380, 51鈥?6 (2008).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 32.Su, Y., Waring, A. J., Ruchala, P. Hong, M. Membrane-bound dynamic structure of an arginine-rich cell-penetrating peptide, the protein transduction domain of HIV TAT, from solid-state NMR. Biochemistry 49, 6009鈥?020 (2010).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 33.McDonald, J. M., Cairns, N. J., Taylor-Reinwald, L., Holtzman, D. Walsh, D. M. The levels of water-soluble and triton-soluble A尾 are increased in Alzheimer鈥檚 disease brain. Brain Res. 1450, 138鈥?47 (2012).CAS聽 PubMed聽 Article聽Google Scholar聽 34.Shenkarev, Z. O. et al. Molecular mechanism of action of 尾-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers. Biochemistry 50, 6255鈥?265 (2011).CAS聽 PubMed聽 Article聽Google Scholar聽 35.Soscia, S. J. et al. The Alzheimer鈥檚 disease-associated amyloid beta-protein is an antimicrobial peptide. PLoS ONE 5, e9505 (2010).ADS聽 PubMed聽 PubMed Central聽 Article聽 CAS聽Google Scholar聽 36.Kumar, D. K. V. et al. Amyloid-尾 peptide protects against microbial infection in mouse and worm models of Alzheimer鈥檚 disease. Sci. Transl. Med. 8, 340ra72鈥?40ra72 (2016).PubMed聽 Article聽 CAS聽Google Scholar聽 37.Li, L., Vorobyov, I., MacKerell, A. D. Allen, T. W. Is arginine charged in a membrane? Biophys. J. 94, L11鈥揕13 (2008).CAS聽 PubMed聽 Article聽Google Scholar聽 38.Moon, C. P. Fleming, K. G. Side-chain hydrophobicity scale derived from transmembrane protein folding into lipid bilayers. Proc. Natl Acad. Sci. USA 108, 10174鈥?0177 (2011).ADS聽 CAS聽 PubMed聽 Article聽Google Scholar聽 39.Walsh, D. M. et al. A facile method for expression and purification of the Alzheimer鈥檚 disease-associated amyloid beta-peptide. FEBS J. 276, 1266鈥?281 (2009).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 40.Assenberg, R. et al. Expression, purification and crystallization of a lyssavirus matrix (M) protein. Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 64, 258鈥?62 (2008).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 41.Serra-Batiste, M. et al. Alzheimer麓s disease-associated A尾42 peptide: expression and purification for NMR structural studies. Curr. Chem. Biol. 11, 50鈥?2 (2017).CAS聽 Article聽Google Scholar聽 42.Kerfah, R. et al. Scrambling free combinatorial labeling of alanine-尾, isoleucine-未1, leucine-proS and valine-proS methyl groups for the detection of long range NOEs. J. Biomol. NMR 61, 73鈥?2 (2015).CAS聽 PubMed聽 Article聽Google Scholar聽 43.Sanders, C. R. S枚nnichsen, F. Solution NMR of membrane proteins: practice and challenges. Magn. Reson. Chem. 44, S24鈥揝40 (2006).CAS聽 PubMed聽 Article聽Google Scholar聽 44.Rossi, P., Xia, Y., Khanra, N., Veglia, G. Kalodimos, C. G. (15)N and (13)C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated, selectively [(1)H,(13)C]-labeled proteins. J. Biomol. NMR 66, 259鈥?71 (2016).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 45.Mayzel, M., Kazimierczuk, K. Orekhov, V. Y. The causality principle in the reconstruction of sparse NMR spectra. Chem. Commun. 50, 8947鈥?950 (2014).CAS聽 Article聽Google Scholar聽 46.Orekhov, V. Y. Jaravine, V. A. Analysis of non-uniformly sampled spectra with multi-dimensional decomposition. Prog. Nucl. Magn. Reson. Spectrosc. 59, 271鈥?92 (2011).CAS聽 PubMed聽 Article聽Google Scholar聽 47.Shao, H., Jao, S., Ma, K. Zagorski, M. G. Solution structures of micelle-bound amyloid beta-(1-40) and beta-(1-42) peptides of Alzheimer鈥檚 disease. J. Mol. Biol. 285, 755鈥?73 (1999).CAS聽 PubMed聽 Article聽Google Scholar聽 48.Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nat. Protoc. 2, 2728鈥?733 (2007).CAS聽 PubMed聽 Article聽Google Scholar聽 49.Rieping, W. et al. ARIA2: automated NOE assignment and data integration in NMR structure calculation. Bioinformatics 23, 381鈥?82 (2007).CAS聽 PubMed聽 Article聽Google Scholar聽 50.Shen, Y. Bax, A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 56, 227鈥?41 (2013).CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 51.Linge, J. P., Williams, M. A., Spronk, C. A. E. M., Bonvin, A. M. J. J. Nilges, M. Refinement of protein structures in explicit solvent. Proteins 50, 496鈥?06 (2003).CAS聽 PubMed聽 Article聽Google Scholar聽 52.Laskowski, R. A., MacArthur, M. W., Moss, D. S. Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283鈥?91 (1993).CAS聽 Article聽Google Scholar聽 53.Vriend, G. WHAT IF: a molecular modeling and drug design program. J. Mol. Graph. 8, 52鈥?6 (2001).Article聽Google Scholar聽 54.Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375鈥揥383 (2007).ADS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 55.Gabelica, V. et al. Recommendations for reporting ion mobility Mass Spectrometry measurements. Mass Spectrom. Rev. https://doi.org/10.1002/mas.21585. (2019)56.Revercomb, H. E. Mason, E. A. Theory of plasma chromatography/gaseous electrophoresis. Anal. Chem. 47, 970鈥?83 (1975).CAS聽 Article聽Google Scholar聽 57.Bush, M. F. et al. Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal. Chem. 82, 9557鈥?565 (2010).CAS聽 PubMed聽 Article聽Google Scholar聽 58.Marklund, E. G., Degiacomi, M. T., Robinson, C. V., Baldwin, A. J. Benesch, J. L. P. Collision cross sections for structural proteomics. Structure 23, 791鈥?99 (2015).CAS聽 PubMed聽 Article聽Google Scholar聽 59.Cadene, M. Chait, B. T. A Robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins. Anal. Chem. 72, 5655鈥?658 (2000).CAS聽 PubMed聽 Article聽Google Scholar聽 60.Jo, S., Kim, T., Iyer, V. G. Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. computational Chem. 29, 1859鈥?865 (2008).CAS聽 Article聽Google Scholar聽 61.McGibbon, R. T. et al. MDTraj: a modern open library for the analysis of molecular dynamics trajectories. Biophys. J. 109, 1528鈥?532 (2015).ADS聽 CAS聽 PubMed聽 PubMed Central聽 Article聽Google Scholar聽 Download referencesAcknowledgementsWe acknowledge Montserrat Serra-Batiste, Mart铆 Ninot-Pedrosa, Margarida Gair铆 and Jes煤s Garc铆a for helpful discussions, sample preparation and NMR data acquisition at earlier stages of the project, James Tolchard for helpful discussions and building octamer models, Marta Vilaseca, Marina Gay, Carol V. Robinson and Michael Landreh for helpful discussions and MS data acquisition at earlier stages of the project, and Oscar Hernandez for help in calibration of CCS measurements. This study was supported by MINECO (SAF2015-68789), the Fondation Recherche M茅dicale (AJE20151234751) and the Counseil R茅gional d鈥橝quitaine Limousin Poitou-Charentes (1R30117-00007559) to N.C. The authors聽G.M and N.C. acknowledge funds from聽Fundaci贸 La Marat贸 de TV3 (20140730). B.B research was supported by the INCEPTION project (ANR-16-CONV-0005). E.T. was supported by the National Institutes of Health (P41-GM104601 and R01-GM123455) and also acknowledges computing resources provided by Blue Waters at National Center for Supercomputing Applications, and Extreme Science and Engineering Discovery Environment XSEDE (grant MCA06N060). V.O. research was supported by the Swedish Research Council Formas (2015-04614). S. Cianferani research was supported by Agence Nationale de la Recherche聽and the French Proteomic Infrastructure (ANR-10-INBS-08-03). N.C., S. Cianferani, T.B. and E.P. acknowledge the support of COST Action (BM1403). E.P. was a PhD fellow funded by MINECO (FPI). T.B. was a PhD fellow funded by Institut de Recherche Servier.Author informationAuthor notesThese authors contributed equally: Sonia Ciudad, Eduard Puig.AffiliationsUniversity of Bordeaux, CBMN (UMR 5248)鈥擟NRS鈥擨PB, Institut Europ茅en de Chimie et Biologie, 2 rue Escarpit, 33600, Pessac, FranceSonia Ciudad,聽Eduard Puig,聽St茅phane Chaignepain聽 聽Nat脿lia CarullaInstitute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028, Barcelona, SpainSonia Ciudad,聽Eduard Puig聽 聽Nat脿lia CarullaDepartament de Qu铆mica Inorg脿nica i Org脿nica, Universitat de Barcelona, Mart铆 i Franqu茅s 1, 08028, Barcelona, SpainEduard PuigLaboratoire de Spectrom茅trie de Masse BioOrganique, Universit茅 de Strasbourg, CNRS UMR7178, IPHC, Strasbourg, FranceThomas Botzanowski聽 聽Sarah CianferaniNIH Center for Macromolecular Modeling and Bioinformatics, Beckman Institute for Advanced Science and Technology, Center for Biophysics and Quantitative Biology and Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USAMoeen Meigooni,聽Andres S. Arango,聽Jimmy Do聽 聽Emad TajkhorshidSwedish NMR Centre, University of Gothenburg, Box 465, 405 30, Gothenburg, SwedenMaxim Mayzel聽 聽Vladislav OrekhovBiochemistry, Molecular and Structural Biology Section, University of Leuven, Celestijnenlaan 200G, 3001, Leuven, BelgiumMariam BayoumiGroningen Biomolecular Sciences Biotechnology Institute, University of Groningen, 9747 AG, Groningen, The NetherlandsGiovanni MagliaDepartment of Chemistry and Molecular Biology, University of Gothenburg, Box 465, 405 30, Gothenburg, SwedenVladislav OrekhovStructural Bioinformatics Unit, Department of Structural Biology and Chemistry, C3BI, Institut Pasteur; CNRS UMR3528; CNRS USR3756, Paris, FranceBenjamin BardiauxAuthorsSonia CiudadView author publicationsYou can also search for this author in PubMed聽Google ScholarEduard PuigView author publicationsYou can also search for this author in PubMed聽Google ScholarThomas BotzanowskiView author publicationsYou can also search for this author in PubMed聽Google ScholarMoeen MeigooniView author publicationsYou can also search for this author in PubMed聽Google ScholarAndres S. ArangoView author publicationsYou can also search for this author in PubMed聽Google ScholarJimmy DoView author publicationsYou can also search for this author in PubMed聽Google ScholarMaxim MayzelView author publicationsYou can also search for this author in PubMed聽Google ScholarMariam BayoumiView author publicationsYou can also search for this author in PubMed聽Google ScholarSt茅phane ChaignepainView author publicationsYou can also search for this author in PubMed聽Google ScholarGiovanni MagliaView author publicationsYou can also search for this author in PubMed聽Google ScholarSarah CianferaniView author publicationsYou can also search for this author in PubMed聽Google ScholarVladislav OrekhovView author publicationsYou can also search for this author in PubMed聽Google ScholarEmad TajkhorshidView author publicationsYou can also search for this author in PubMed聽Google ScholarBenjamin BardiauxView author publicationsYou can also search for this author in PubMed聽Google ScholarNat脿lia CarullaView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsN.C. designed and coordinated the project. S.C. prepared all the NMR samples and analyzed all the NMR spectra. M.M. and V.O. acquired most of the NMR experiments required for this study. B.B. calculated the structure of the A尾(1-42) tetramer from NMR restraints and contributed to analyze CCS measurements. E.P. prepared all the MS samples and analysed them by SEC and SDS-PAGE. E.P., T.B., and S.Cianferani acquired and analysed the samples by SEC/IM-MS. E.P. and S.Chaignepain acquired and analysed the samples by MALDI-TOF. G.M. and M.B. performed and analysed electrical recordings using lipid bilayers. E.T., M.Meigooni, A.S.A. and J. D. carried out and analysed gas phase and detergent and membrane simulations. N.C. wrote the paper with input and contributions from all authors.Corresponding authorCorrespondence to Nat脿lia Carulla.Ethics declarations Competing interests The authors declare no competing interests. Additional informationPeer review information Nature Communications thanks Charles Sanders, Edward Stern, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.Publisher鈥檚 note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary information CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.