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Molecular insights into the surface-catalyzed secondary nucleation of amyloid-β40 (Aβ40) by the peptide fragment Aβ16–22

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Science Advances  21 Jun 2019:
Vol. 5, no. 6, eaav8216
DOI: 10.1126/sciadv.aav8216
  • Fig. 1 Co-aggregation of Aβ16–22 and Aβ40 results in accelerated aggregation kinetics for Aβ40.

    (A) Primary sequence of Aβ16–22 and Aβ40, including the groups at each terminus. The central recognition motif KLVFF is highlighted in purple. (B) ThT fluorescence assays showing that the aggregation rate of Aβ40 increases as the ratio of Aβ16–22 to Aβ40 is increased (with the total peptide concentration held constant at 40 μM). (C) Simulation snapshots of the aggregation of six Aβ40 monomers into a β-sheet–rich hexamer at an Aβ40 concentration of 5 mM. At the start of the simulation (0 μs), all the peptides are in random coils, but as the simulation progresses, they aggregate into antiparallel, in-register β sheets (104 μs). This oligomer then unfolds, losing some of its β-sheet structure (230 μs) before a rearrangement in which the β sheets rearrange, forming a stable fibril, with each Aβ40 peptide containing three β strands (621 μs) engaged in parallel intermolecular hydrogen bonding.

  • Fig. 2 Aggregation kinetics of Aβ16–22 are unaffected by the presence of Aβ40.

    (A) Schematic showing the principle behind the fluorescence quenching assay used to determine the aggregation rate of Aβ16–22. (B) As self-assembly occurs, the TAMRA-labeled peptides [40 μM total peptide containing 5% (w/w) TAMRA-Ahx-Aβ16–22] are sequestered into the fibril structure. This brings the fluorophores into proximity, resulting in fluorescence quenching. (C) Aggregation of Aβ16–22 [containing 5% (w/w) TAMRA-Ahx-Aβ16–22] and Aβ40 at a 1:1 mol/mol ratio (40 μM total peptide concentration). A single transient, which is the median of three replicates measured, is shown. (D and E) Sedimentation and separation of the pellet and supernatant of the 1:1 mixed system and analysis of the fractions using ESI-MS after 1 hour indicate that Aβ40 is present in the (D) supernatant and in only very small amounts within the (E) pellet. (F) Under these conditions, fibrils of Aβ16–22 are present after 5 min of incubation. Scale bars, 500 nm.

  • Fig. 3 16–22 can interact with Aβ40 monomers and dimers.

    (A) Native ESI-IMS-MS drift-scope images of Aβ40 indicate the presence of multiple oligomeric species of Aβ40 (white numbers). (B) When mixed at a 1:1 mol/mol ratio with Aβ16–22 (yellow numbers), a number of heteromeric species are observed (light blue numbers) immediately following mixing. The oligomer size is given (1, 2, 3, etc.), with the charge state in superscript. (C) DMD simulation showing the percent β sheet formed by Aβ40 during aggregation in the absence (black) or presence (red) of Aβ16–22. (D) Energy contact map between one monomer of Aβ16–22 and one of Aβ40 scaled by energy (bar shown alongside), showing that residues 17 to 20 (LVFF) and 31 to 34 (IIGL) form the strongest interactions. (E) Co-aggregation can have differing effects on the primary nucleation of each peptide, depending on whether the mixed oligomers formed can progress to form mixed fibrils or are off-pathway and take no further part in the aggregation reaction. Circles represent monomers and blocks represent fibrils, with Aβ16–22 and Aβ40 in red and blue, respectively. Adapted from (24).

  • Fig. 4 16–22 fibrils increase the aggregation rate of Aβ40 to a greater extent than Aβ16–22 monomers.

    (A) Increased concentrations (% w/w) of Aβ16–22 fibrils were added to Aβ40 monomers (as shown in the key), and the aggregation rate was measured by ThT fluorescence. (B) Direct comparison of the effect of Aβ16–22 monomers (i.e., taken straight from a DMSO stock) and Aβ16–22 fibrils on Aβ40 aggregation. (C) Effect of sonicating the Aβ16–22 fibrils on the Aβ40 aggregation rate shows little effect compared with the data shown in (A) (see text for details). (D) Plots of the percent β sheet formed by Aβ40 in the absence (blue) or presence of preformed two (black), three (red), or four (green) β-sheet Aβ16–22, determined using DMD, showing that an increased Aβ16–22 fibril size increases the rate of Aβ40 aggregation. (E) During co-aggregation experiments, both elongation and surface-catalyzed mechanisms can occur; each has a different effect on the rate of assembly of each peptide (the same notation is used as in Fig. 3E, with circles representing monomers, blocks representing fibrils, and Aβ16–22 and Aβ40 in red and blue, respectively). Adapted from (24).

  • Fig. 5 16–22and Aβ40do not co-assemble during co-aggregation.

    Negative-stain TEM analysis of Aβ40 incubated for 24 hours in the (A) absence or (B) presence of Aβ16–22. Scale bars, 200 nm. (C) PIC of mixtures of diazirine-labeled Aβ16–22 (Aβ*16–22) and Aβ40 incubated for 24 hours and then irradiated for 30 s. Only homomolecular Aβ16–22 cross-links are observed, indicating that the fibrils are not copolymerized at the end of the reaction (the inset depicts the mechanism of PIC of the diazirine group. (D) DMD simulation snapshots of co-aggregation of Aβ40 (blue) and Aβ16–22 (red) indicate that separate homomolecular oligomers are formed at t = 202 μs.

  • Fig. 6 16–22fibrils catalyze Aβ40assembly through secondary surface nucleation.

    (A) Simulation snapshots of the process by which Aβ16–22 fibrils (red) increase the aggregation rate of Aβ40 (blue) through a surface-catalyzed secondary nucleation. (B) A schematic description of the mechanism is also included, with Aβ40 in blue and Aβ16–22 in red.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaav8216/DC1

    General materials and methods for organic synthesis

    Synthesis of N-Fmoc–protected TFMD-Phe

    General materials and methods for Aβ16–22 solid-phase peptide synthesis

    General materials and methods for HPLC purification

    Analytical MS and HPLC data for synthetic peptides

    General materials and methods for recombinant peptide synthesis

    Additional characterization and analyses

    CCS analysis of Aβ40 in the presence and absence of Aβ16–22

    Scheme S1. Synthesis of TFMD-Phe.

    Fig. S1. HRMS and analytical HPLC traces of Aβ16–22 and its variants.

    Fig. S2. SEC trace of Aβ40 indicates that there is a single peak, and ESI-IMS-MS indicates that in the gas phase Aβ40 is largely monomeric.

    Fig. S3. Supplementary ThT data.

    Fig. S4. Supplementary negative-stain TEM images.

    Fig. S5. Analysis of the CCS values for Aβ40 in the absence or presence of Aβ16–22 over different IMS experiments.

    Fig. S6. PIC analysis of 1:1 Aβ*16–22/Aβ40 at 5 min and 24 hours.

    Fig. S7. Plot of the average number of hydrogen bonding and side chain–side chain.

    Table S1. The expected and observed m/z values for monomeric and oligomeric Aβ40 in isolation and in the presence of a 1:1 ratio of Aβ16–22.

    Table S2. Assignments of each of the major peaks observed in fig. S6A.

    Data file S1. MD snapshots as pdb files Fig. 1 t = 0.

    Data file S2. MD snapshots as pdb files Fig. 1 t = 104.

    Data file S3. MD snapshots as pdb files Fig. 1 t = 230.

    Data file S4. MD snapshots as pdb files Fig. 1 t = 621.

    Data file S5. MD snapshots as pdb files Fig. 6 t = 0.29.

    Data file S6. MD snapshots as pdb files Fig. 6 t = 1.16.

    Data file S7. MD snapshots as pdb files Fig. 6 t = 1.93.

    Data file S8. MD snapshots as pdb files Fig. 6 t = 7.7.

    Data file S9. MD snapshots as pdb files Fig. 6 t = 29.

    Data file S10. MD snapshots as pdb files Fig. 6 t = 77.7.

  • Supplementary Materials

    The PDF file includes:

    • General materials and methods for organic synthesis
    • Synthesis of N-Fmoc–protected TFMD-Phe
    • General materials and methods for Aβ16–22 solid-phase peptide synthesis
    • General materials and methods for HPLC purification
    • Analytical MS and HPLC data for synthetic peptides
    • General materials and methods for recombinant peptide synthesis
    • Additional characterization and analyses
    • CCS analysis of Aβ40 in the presence and absence of Aβ16–22
    • Scheme S1. Synthesis of TFMD-Phe.
    • Fig. S1. HRMS and analytical HPLC traces of Aβ16–22 and its variants.
    • Fig. S2. SEC trace of Aβ40 indicates that there is a single peak, and ESI-IMS-MS indicates that in the gas phase Aβ40 is largely monomeric.
    • Fig. S3. Supplementary ThT data.
    • Fig. S4. Supplementary negative-stain TEM images.
    • Fig. S5. Analysis of the CCS values for Aβ40 in the absence or presence of Aβ16–22 over different IMS experiments.
    • Fig. S6. PIC analysis of 1:1 Aβ*16–22/Aβ40 at 5 min and 24 hours.
    • Fig. S7. Plot of the average number of hydrogen bonding and side chain–side chain.
    • Table S1. The expected and observed m/z values for monomeric and oligomeric Aβ40 in isolation and in the presence of a 1:1 ratio of Aβ16–22.
    • Table S2. Assignments of each of the major peaks observed in fig. S6A.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (.pdb format). MD snapshots as pdb files Fig. 1 t = 0.
    • Data file S2 (.pdb format). MD snapshots as pdb files Fig. 1 t = 104.
    • Data file S3 (.pdb format). MD snapshots as pdb files Fig. 1 t = 230.
    • Data file S4 (.pdb format). MD snapshots as pdb files Fig. 1 t = 621.
    • Data file S5 (.pdb format). MD snapshots as pdb files Fig. 6 t = 0.29.
    • Data file S6 (.pdb format). MD snapshots as pdb files Fig. 6 t = 1.16.
    • Data file S7 (.pdb format). MD snapshots as pdb files Fig. 6 t = 1.93.
    • Data file S8 (.pdb format). MD snapshots as pdb files Fig. 6 t = 7.7.
    • Data file S9 (.pdb format). MD snapshots as pdb files Fig. 6 t = 29.
    • Data file S10 (.pdb format). MD snapshots as pdb files Fig. 6 t = 77.7.

    Files in this Data Supplement:

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