Amyloid Fibril Formation by Aβ16-22, a Seven-Residue Fragment of the Alzheimer's β-Amyloid Peptide, and Structural Characterization by Solid State NMR†
TL;DR: One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit 13C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils.
Abstract: The seven-residue peptide N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH(2), called A beta(16-22) and representing residues 16-22 of the full-length beta-amyloid peptide associated with Alzheimer's disease, is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements confirm that these are amyloid fibrils. The peptide conformation and supramolecular organization in A beta(16-22) fibrils are investigated by solid state (13)C NMR measurements. Two-dimensional magic-angle spinning (2D MAS) exchange and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) measurements indicate a beta-strand conformation of the peptide backbone at the central phenylalanine. One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit (13)C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils. Two-dimensional (13)C-(13)C chemical shift correlation spectroscopy permits a nearly complete assignment of backbone and side chain (13)C NMR signals and indicates that the beta-strand conformation extends across the entire hydrophobic segment from Leu17 through Ala21. (13)C multiple-quantum (MQ) NMR and (13)C/(15)N rotational echo double-resonance (REDOR) measurements indicate an antiparallel organization of beta-sheets in the A beta(16-22) fibrils. These results suggest that the degree of structural order at the molecular level in amyloid fibrils can approach that in peptide or protein crystals, suggest how the supramolecular organization of beta-sheets in amyloid fibrils can be dependent on the peptide sequence, and illustrate the utility of solid state NMR measurements as probes of the molecular structure of amyloid fibrils. A beta(16-22) is among the shortest fibril-forming fragments of full-length beta-amyloid reported to date, and hence serves as a useful model system for physical studies of amyloid fibril formation.
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TL;DR: There is increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity, leading to approaches toward rational therapeutics.
Abstract: Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and prion diseases are increasingly being realized to have common cellular and molecular mechanisms including protein aggregation and inclusion body formation. The aggregates usually consist of fibers containing misfolded protein with a beta-sheet conformation, termed amyloid. There is partial but not perfect overlap among the cells in which abnormal proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves. For several diseases, genetic variants assist in explaining the pathogenesis of the more common sporadic forms and developing mouse and other models. There is now increased understanding of the pathways involved in protein aggregation, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity. These are leading to approaches toward rational therapeutics.
2,926 citations
Journal Article•
TL;DR: The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves as discussed by the authors.
2,416 citations
TL;DR: A structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimer's disease (Aβ1–40) is presented, based on a set of experimental constraints from solid state NMR spectroscopy and incorporates the cross-β structural motif established by x-ray fiber diffraction.
Abstract: We present a structural model for amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-beta structural motif established by x-ray fiber diffraction and satisfies constraints on Abeta(1-40) fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Abeta(1-40) are structurally disordered in the fibrils. Residues 12-24 and 30-40 adopt beta-strand conformations and form parallel beta-sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the peptide backbone that brings the two beta-sheets in contact through sidechain-sidechain interactions. A single cross-beta unit is then a double-layered beta-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-beta units with their hydrophobic faces juxtaposed.
1,842 citations
TL;DR: Solid-state nuclear magnetic resonance measurements on fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease are described and full molecular models using restrained molecular dynamics simulations and restrained energy minimization are constructed.
Abstract: We describe solid-state nuclear magnetic resonance (NMR) measurements on fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimer's disease (Aβ1-40) that place constraints on the identity and symmetry of contacts between in-register, parallel β-sheets in the fibrils. We refer to these contacts as internal and external quaternary contacts, depending on whether they are within a single molecular layer or between molecular layers. The data include (1) two-dimensional 13C-13C NMR spectra that indicate internal quaternary contacts between side chains of L17 and F19 and side chains of I32, L34, and V36, as well as external quaternary contacts between side chains of I31 and G37; (2) two-dimensional 15N-13C NMR spectra that indicate external quaternary contacts between the side chain of M35 and the peptide backbone at G33; (3) measurements of magnetic dipole−dipole couplings between the side chain carboxylate group of D23 and the side chain amine group of K28 that indicate salt bridge interact...
1,048 citations
Cites background from "Amyloid Fibril Formation by Aβ16-22..."
...the primary stabilizing interactions in Aβ1-40 fibrils and in fibrils formed by certain other peptides (8, 10, 37, 41), may account for the polymorphism of Aβ1-40 fibrils....
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...Solid state nuclear magnetic resonance (NMR) (8-19), hydrogen/deuterium (H/D) exchange (14, 20-27), proline-scanning mutagenesis (28, 29), electron paramagnetic resonance (EPR) (30-32), and infrared and Raman spectroscopies (33, 34) have been applied to the problem of secondary structure determination....
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...Solid state NMR (7, 8, 11, 15, 16, 19, 35-44) and EPR (30-32, 45, 46) measurements have been particularly useful in experimental determinations of tertiary structure....
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TL;DR: The π‐stacking hypothesis suggests a new approach to understanding the self‐assembly mechanism that governs amyloid formation and indicates possible ways to control this process.
Abstract: Amyloid fibril formation is assumed to be the molecular basis for a variety of diseases of unrelated origin. Despite its fundamental clinical importance, the mechanism of amyloid formation is not fully understood. When we analyzed a variety of short functional fragments from unrelated amyloid-forming proteins, a remarkable occurrence of aromatic residues was observed. The finding of aromatic residues in diverse fragments raises the possibility that pi-pi interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation. This is in line with the well-known central role of pi-stacking interactions in self-assembly processes in the fields of chemistry and biochemistry. We speculate that the stacking interactions may provide energetic contribution as well as order and directionality in the self-assembly of amyloid structures. Experimental data regarding amyloid formation and inhibition by short peptide analogs also support our hypothesis. The pi-stacking hypothesis suggests a new approach to understanding the self-assembly mechanism that governs amyloid formation and indicates possible ways to control this process.
1,022 citations
References
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TL;DR: An analysis of the 1H nuclear magnetic resonance chemical shift assignments and secondary structure designations for over 70 proteins has revealed some very strong and unexpected relationships.
1,862 citations
TL;DR: Using intense synchrotron sources, it is observed that six different ex vivo amyloid fibrils and two synthetic fibril preparations all gave similar high-resolution X-ray fibre diffraction patterns, consistent with a helical array of beta-sheets parallel to the fibre long axis, with the strands perpendicular to this axis, which confirms that amyloidsfibrils comprise a structural superfamily and share a common protofilament substructure.
1,648 citations
TL;DR: In this article, general integral and series expressions are derived for the intensities of sidebands observed in the magic angle spectra of inhomogeneously broadened I = 1/2 systems.
Abstract: General integral and series expressions are derived for the intensities of sidebands observed in the magic angle spectra of inhomogeneously broadened I=1/2 systems The expressions are evaluated for a wide range of shift parameters and the results used to construct graphical and numerical methods for extracting the principal values of chemical shift tensors from the intensities of just a few sidebands The methods are illustrated by application to 31P spectra of barium diethyl phosphate The results agree well with previous single crystal measurements
1,476 citations
TL;DR: Results indicate that the length of the hydrophobic carboxyl termini is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.
1,061 citations
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