Atomic structure and assembly of an amyloid fibril (Proc. Natl. Acad. Sci. USA 110, 5468, 2013)

We have published the structure of cross-β amyloid fibrils formed by TTR in the Proceedings of the National Academy of Sciences ().

The work was led by Dr. Anthony Fitzpatrick and the authors include three generations of my colleagues, directed and mentored by Robert Griffin and Christopher Dobson. The publication has been reviewed in Physics Today (download: Atomic and hierarchical structure of an amyloid fibril | Physics Update – Physics Today). It is a prototypical example of the integration of multiple biophysical techniques to approach mesoscale structure.

Five diverse biophysical techniques were integrated to determine unambiguously the structures of each of the motifs that make up the TTR(105–115) amyloid fibrils. Spanning five orders of magnitude, the overlapping length scales of MAS NMR (0.1–10 Å), X-ray diffraction (3–100 Å), cryo-EM (8–1,000 Å), AFM (30–1,000 Å), and STEM (80–1,000 Å) enabled us to derive self-consistent, high-precision structural restraints on the secondary (β-strand and -sheet; distance restraints of <6 Å), tertiary (protofilament; distance restraints of 4.5–37 Å), and quaternary structure (filament and fibril; distance restraints of 16–1,000 Å) of the TTR(105–115) amyloid fibrils. (A) Histogram of STEM MPL measurements of TTR(105–115) fibrils, which reveals three populations of fibrils, with a best fit (gray solid line) being the sum of three Gaussian curves with values of 2.5 ± 0.3 kDa/Å (orange solid line), 3.3 ± 0.3 kDa/Å (yellow solid line), and 4.1 ± 0.3 kDa/Å (purple solid line). The orange, yellow, and purple dashed lines refer to the number of TTR(105–115) peptides per 4.67-Å repeat in the doublet (8 peptides), triplet (12 peptides), and quadruplet (16 peptides) fibrils, respectively. (B) Comparison of the high-resolution experimental X-ray diffraction pattern from TTR(105–115) fibrils (34) (Left) and the simulated X-ray diffraction pattern for TTR(105–115) fibrils (Right). The fibril axis is vertical, with the incident beam directed orthogonally to this axis. The meridional reflection at 4.67 Å and the equatorial reflection at 8.86 Å are characteristic of cross-β structure. (C) High-resolution AFM image of fibrils (pink and purple) and filaments (green) formed by TTR(105–115). (Scale bar, 1 μm.) Fibrils (pink and purple) have heights ranging from 70 to 160 Å and pitches of 950 ± 100 Å. The filament has an average height of 38.7 ± 4.4 Å. (D) Hierarchy of atomic-resolution motifs involved in the self-assembly of the amyloid fibrils and their polymorphism.

Five diverse biophysical techniques were integrated to determine unambiguously the structures of each of the motifs that make up the TTR(105–115) amyloid fibrils. Spanning five orders of magnitude, the overlapping length scales of MAS NMR (0.1–10 Å), X-ray diffraction (3–100 Å), cryo-EM (8–1,000 Å), AFM (30–1,000 Å), and STEM (80–1,000 Å) enabled us to derive self-consistent, high-precision structural restraints on the structure.

Close-up view of the MAS NMR atomic-resolution structure of the triplet fibril fitted into the cryo-EM reconstruction (Center). The background image of the fibril (Left) was taken using TEM. (Scale bar, 50 nm.) The fibril surfaces (Right) are shown at 1.0σ (white) and 2.2σ (yellow) above the mean density, respectively, and the constituent β-sheets are shown in a ribbon representation; oxygen, carbon, and nitrogen atoms are shown in red, gray, and blue, respectively.

Close-up view of the MAS NMR atomic-resolution structure of the triplet fibril fitted into the cryo-EM reconstruction (Center). The background image of the fibril (Left) was taken using TEM. (Scale bar, 50 nm.) The fibril surfaces (Right) are shown at 1.0σ (white) and 2.2σ (yellow) above the mean density, respectively, and the constituent β-sheets are shown in a ribbon representation; oxygen, carbon, and nitrogen atoms are shown in red, gray, and blue, respectively.

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Atomic structure and hierarchical assembly of a cross-β amyloid fibril

Abstract

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

 

 

 

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