Spectroscopic Signature for Stable β-Amyloid Fibrils versus β-Sheet-Rich Oligomers

Abstract

We use two-dimensional IR (2D IR) spectroscopy to explore fibril formation for the two predominant isoforms of the β-amyloid (Aβ_1-40 and Aβ_1-42) protein associated with Alzheimer's disease. Two-dimensional IR spectra resolve a transition at 1610 cm^-1 in Aβ fibrils that does not appear in other Aβ aggregates, even those with predominantly β-sheet-structure-like oligomers. This transition is not resolved in linear IR spectroscopy because it lies under the broad band centered at 1625 cm^-1, which is the traditional infrared signature for amyloid fibrils. The feature is prominent in 2D IR spectra because 2D lineshapes are narrower and scale nonlinearly with transition dipole strengths. Transmission electron microscopy measurements demonstrate that the 1610 cm^-1 band is a positive identification of amyloid fibrils. Sodium dodecyl sulfate micelles that solubilize and disaggregate preaggregated Aβ samples deplete the 1625 cm^-1 band but do not affect the 1610 cm^-1 band, demonstrating that the 1610 cm^-1 band is due to very stable fibrils. We demonstrate that the 1610 cm^-1 transition arises from amide I modes by mutating out the only side-chain residue that could give rise to this transition, and we explore the potential structural origins of the transition by simulating 2D IR spectra based on Aβ crystal structures. It was not previously possible to distinguish stable Aβ fibrils from the less stable β-sheet-rich oligomers with infrared light. This 2D IR signature will be useful for Alzheimer's research on Aβ aggregation, fibril formation, and toxicity.

Figure 1

Two-dimensional IR spectra of 1 mM Aβ aggregated for 30 min, 1 day, and 7 days, diagonal slices through the fundamental transition, and representative TEM images for the following samples: (a) Aβ_1–40 in pD 7.5 20 mM Tris buffer, (b) Aβ_1–42 in pD 7.5 20 mM Tris buffer, (c) Aβ_1–40 in pD 2.0 0.04% NaN₃ solution, and (d) Aβ_1–42 in pD 2.0 0.04% NaN₃ solution.

Figure 2

Two-dimensional IR spectra and diagonal slices of 1 mM Aβ_1–42,R5G collected after 6 days of aggregation in pD 2.0 0.04% NaN₃ solution and after 14 days of aggregation in pD 7.5 20 mM Tris buffer and corresponding TEM images. Fibril formation in the pD 7.5 buffer took longer than in the pD 2.0 buffer. Similar to the 2D IR spectra of Aβ_1–40 and Aβ_1–42 fibrils shown in Figure 1, a transition is observed at ca. 1610 cm⁻¹.

Figure 3

(a) Two-dimensional IR spectra and diagonal slices of 1 mM Aβ_1–40 and Aβ_1–42 reconstituted directly into 100 mM SDS. (b) Two-dimensional IR spectra and diagonal slices of Aβ_1–40 aggregated for 30 min in pD 7.5 Tris buffer and 30 min after the addition of an equal volume 100 mM SDS. (c) Kinetic traces from rapid scan 2D IR in the β-sheet and α-helical regions for Aβ_1–42 aggregated for 1 day in pD 2.0 NaN₃ solution, and diagonal slices at various disaggregation times scaled to the maximum intensity. (d) Two-dimensional IR spectra of Aβ_1–42 aggregated for 6 weeks in pD 2.0 NaN₃ solution and 3 weeks after the addition of an equal volume 100 mM SDS.

Figure 4

(a) ssNMR structure of an Aβ fibril obtained from PDB structure 2mxu. (b) Atomic structures of Aβ_35–42 fragments from PDB file 2y3k from which β-sheets were generated as described in the text. (c) The 2D IR spectrum simulated as a mixture of the two structures from panel (b) added in a 1:4 ratio to simulate the spectrum observed experimentally. Note that the local mode transition frequency was shifted to best match the experimental data, though the difference in frequency is unaffected by this shift. Details of the calculations are given in Materials and Methods section.