Solid-state NMR studies of amyloid fibril structure

Abstract

Current interest in amyloid fibrils stems from their involvement in neurodegenerative and other diseases and from their role as an alternative structural state for many peptides and proteins. Solid-state nuclear magnetic resonance (NMR) methods have the unique capability of providing detailed structural constraints for amyloid fibrils, sufficient for the development of full molecular models. In this article, recent progress in the application of solid-state NMR to fibrils associated with Alzheimer's disease, prion fibrils, and related systems is reviewed, along with relevant developments in solid-state NMR techniques and technology.

Figure 1

Negatively-stained TEM images of amyloid fibrils. (a) “Striated ribbon” Aβ_1-40 fibrils. (b) “Twisted pair” Aβ_1-40 fibrils. (c) Brain-seeded Aβ_1-40 fibrils. (d) Amylin fibrils. (e) Sup35NM fibrils. (f) HET-s_218-289 fibrils. White scale bars are 100 nm. (g,h,i) Schematic representations of cross-β structures formed by parallel β-sheets, antiparallel β-sheets, and a β-helix, respectively.

Figure 2

Molecular structural models for “striated ribbon” (a) and “twisted pair” (b) Aβ_1-40 fibrils, developed primarily from solid state NMR data with additional constraints from electron microscopy. These models include residues 9-40, with hydrophobic, negatively charged, postively charged (including His), and polar (including Gly) residues colored green, red, blue, and magenta, respectively. Both models are viewed down the long axis of the fibril, with three repeats shown. The full Aβ_1-40 sequence is shown below.

Figure 3

MPL data for Aβ_1-40 fibrils. (a,c) Dark-field TEM images of unstained Aβ_1-40 fibrils with “twisted pair” and “striated ribbon” morphologies, respectively. Arrows indicate tobacco mosaic virus (TMV) rods that serve as intensity calibration standards. Rectangles enclose fibril segments with the indicated MPL values in kDa/nm. White scale bars are 200 nm. (b,d) MPL histograms obtained from multiple dark-field images. Heavy dashed lines indicate the predicted MPL values for the 3-fold symmetric and 2-fold symmetric structural models in Figure 2, which are comprised of three and two cross-β units, respectively. Solid lines are multiple-Gaussian fits. Minor populations comprised of a single cross-β unit appear at MPL ≈ 9 kDa/nm, possibly arising from disruption of the fibril structures during TEM sample preparation or measurement.

Figure 4

2D solid state NMR spectra of uniformly ¹⁵N,¹³C-labeled Aβ_1-40 fibrils prepared by seeding with amyloid extracted from occipital lobe tissue of a deceased Alzheimer’s disease patient. Assignments of selected crosspeaks to specific residues are indicated. (a) 2D ¹³C-¹³C NMR spectrum, obtained in a 14.1 T magnetic field with 13.6 kHz MAS, using a 2.94 ms finite-pulse radio-frequency-driven recoupling (fpRFDR) sequence (117) for spin polarization transfer in the mixing period between the two spectral dimensions. (b) 2D ¹⁵N-¹³C spectrum, obtained with frequency-selective ¹⁵N-¹³C cross-polarization followed by fpRFDR in the mixing period.

Figure 5

Experimental determination of β-sheet organization in wild-type and D23N mutant Aβ_1-40 fibrils with ¹³C and ¹⁵N labels at specific sites. (a) Measurements of intermolecular ¹³C-¹³C dipole-dipole couplings in wild-type fibrils using the PITHIRDS-CT technique (98). Squares, circles, and triangles are data for A30 methyl distances in “striated ribbon” fibrils, A30 methyl distances in “twisted pair” fibrils, and M35 methyl distances in “twisted pair” fibrils. Decay of ¹³C NMR signals on the 30 ms time scale results from ~5 Å intermolecular ¹³C-¹³C distances, indicating an in-register parallel β-sheet structure. (b) Measurements of intermolecular couplings among A21 methyl carbons in D23N-Aβ_1-40 fibrils containing parallel (triangles) and antiparallel (circles and squares) β-sheet structures. (c) Measurements of intermolecular ¹⁵N-¹³C dipole-dipole couplings between the backbone amide nitrogen of L17 and the methyl carbon of A21 in D23N-Aβ_1-40 fibrils containing parallel (triangles) and antiparallel (circles and squares) β-sheet structures, using the REDOR technique (106). Build-up of the normalized REDOR difference signal ΔS/S0 on the 50 ms time scale indicates ~5 Å L17-A21 distances, consistent with an antiparallel β-sheet structure with 17+k ↔ 21-k hydrogen bond registry. (d) Schematic representation of cross-β units in which the two hydrophobic segments of Aβ_1-40 (dark and light green arrows) form antiparallel (top) and parallel (bottom) β-sheets. Favorable hydrophobic interactions can occur in both structures.

Figure 6

Comparison of the HET-s_218-289 prion fibril structure (a,b) with that of the β-helical protein Bordetella pertussis pertactin (c,d), based on Protein Data Bank files 2RNM and 1DAB, respectively. Structures are viewed perpendicular (a,c) and parallel (b,d) to the long axis of the fibril or β-helix. For HET-s_218-289, five repeats of residues 226-278 are shown, with each repeat forming two “rungs” of the β-helix. For pertactin, residues 1-285 is shown. Hydrophobic, negatively charged, postively charged (including His), and polar (including Gly) residues are colored green, red, blue, and magenta, respectively.