High resolution structural characterization of Aβ42 amyloid fibrils by magic angle spinning NMR

Abstract

The presence of amyloid plaques composed of amyloid beta (Aβ) fibrils is a hallmark of Alzheimer's disease (AD). The Aβ peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aβ1-40 and Aβ1-42, respectively. While there have been numerous reports that structurally characterize fibrils of Aβ1-40, very little is known about the structure of amyloid fibrils of Aβ1-42, which are considered the more toxic alloform involved in AD. We have prepared isotopically (13)C/(15)N labeled AβM01-42 fibrils in vitro from recombinant protein and examined their (13)C-(13)C and (13)C-(15)N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aβ fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of AβM01-42 fibrils utilizing (13)C and (15)N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D (13)C-(13)C, (13)C-(15)N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16-42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 β-strands are present in the fibril's secondary structure.

Figure 1

Negative stain transmission micrograph of Aβ_M01–42 fibrils.

Figure 2

1D MAS NMR spectra of Aβ_M01–42 recorded at ω_0H/2π = 800 MHz at 277 K and ω_r/2π = 20 kHz with 83 kHz ¹H decoupling during acquisition. (a) Cross-polarization 1D ¹³C spectrum recorded with 512 transients. (b) ¹³C-INEPT spectrum recorded with 1024 transients. (c) Cross-polarization 1D ¹⁵N spectrum recorded with 512 transients.

Figure 3

(a) 1.6 ms mixing RFDR spectrum recorded at 800 MHz, ω_r/2π = 20 kHz, VT gas regulated to 277 K with 83 kHz CW ¹H decoupling during evolution, and 83 kHz TPPM ¹H decoupling during acquisition. (b) 1.6 ms mixing ZF-TEDOR spectrum recorded at 800 MHz, ω_r/2π = 20 kHz, VT gas regulated to 277 K with 83 kHz TPPM during acquisition.

Figure 4

Representative strip plot of NCOCX (green), CONCA (red), and NCACX (blue) spectra (recorded at 750 and 800 MHz, respectively). ω_r/2π = 12.5 kHz, T = 277 K, τ_mix(DARR) = 80 ms. A 83 kHz ¹H decoupling field was applied during acquisition.

Figure 5

2D ¹³C–¹³C-TOBSY recorded at T = 277 K, ω_0H/2π = 800 MHz, ω_r/2π = 20 kHz, and τ_mix(TOBSY) = 9.6 ms. A 83 kHz ¹H decoupling field was applied during acquisition.

Figure 6

2D ¹³C–¹³C MAS spectrum of Aβ_M01–42 fibrils using DARR mixing recorded at a field strength corresponding to ω_0H/2π = 800 MHz, T = 277 K and ω_r/2π = 20 kHz. τ_mix = 80 ms, with a 83 kHz ¹H decoupling field applied during acquisition.

Figure 7

Schematic showing the amino acids and side chains with the side chains filled in. Red circles correspond to assigned ¹³C’s, blue circles correspond to assigned ¹⁵N’s, and gray circles correspond to resonances that are not observed.

Figure 8

Predicted ψ and φ angles using TALOS. Shaded blue regions indicate predicted β-strands. On the top of the figure the black boxes indicate the location of the beta strands, the solid black line indicate regions that are in loop or random coil conformations and the dashed line indicates regions which have not yet been assigned.

Figure 9

Comparison of the locations of predicted β-strands in various Aβ fibrils. Here, the comparison is limited to NMR chemical shift data (see Figure S1).