Sensitivity-Enhanced Solid-State NMR Detection of Structural Differences and Unique Polymorphs in Pico- to Nanomolar Amounts of Brain-Derived and Synthetic 42-Residue Amyloid-β Fibrils

Abstract

Amyloid-β (Aβ) fibrils in neuritic plaques are a hallmark of Alzheimer’s disease (AD). Since the 42-residue Aβ (Aβ42) fibril is the most pathogenic among different Aβ species, its structural characterization is crucial to our understanding of AD. While several polymorphs have been reported for Aβ40, previous studies of Aβ42 fibrils prepared at neutral pH detected essentially only one structure, with an S-shaped β-sheet arrangement (e.g., Nat. Struct. Mol. Biol.201522499). Herein, we demonstrate the feasibility of characterizing the structure of trace amounts of brain-derived and synthetic amyloid fibrils by sensitivity-enhanced 1H-detected solid-state NMR (SSNMR) under ultrafast magic angle spinning. By taking advantage of the high sensitivity of this technique, we first demonstrate its applicability for the high-throughput screening of trace amounts of selectively 13C- and 15N-labeled Aβ42 fibril prepared with ∼0.01% patient-derived amyloid (ca. 4 pmol) as a seed. The comparison of 2D 13C/1H SSNMR data revealed marked structural differences between AD-derived Aβ42 (∼40 nmol or ∼200 μg) and synthetic fibrils in less than 10 min, confirming the feasibility of assessing the fibril structure from ∼1 pmol of brain amyloid seed in ∼2.5 h. We also present the first structural characterization of synthetic fully protonated Aβ42 fibril by 1H-detected 3D and 4D SSNMR. With procedures assisted by automated assignments, main-chain resonance assignments were completed for trace amounts (∼42 nmol) of a fully protonated amyloid fibril in the 1H-detection approach. The results suggest that this Aβ42 fibril exhibits a novel fold or polymorph structure.

1 H detection and dynamic nuclear polarization–enhanced NMR of Aβ 1-42 fibrils

“…Recently, Wickramasinghe et al. identified a new form of Aβ 1-42 using 1 H detection on brain-seeded fibrils ( 58 ). Likewise, a very different structure of Aβ 1-42 was published by Gremer et al.…”

1H-Detected Biomolecular NMR under Fast Magic-Angle Spinning

“…Experimental conditions influence the formation of specific polymorphs and the sample homogeneity in general . Preformed seeds can be used to accelerate fibril formation and drive it toward a specific polymorph of particular interest . Fibrils are packed in rotors as hydrated protein aggregates.…”

“…Good spectral dispersion, limited overlap and most importantly very long coherence lifetimes allowed the assignment of backbone and side-chain signals (≃92% completeness) within the fibril core by the use of a small set of three-dimensional experiments, recorded within a few days on 0.5 mg of sample . In parallel, Ishii and co-workers explored the feasibility to characterize the structure of synthetic Aβ42 amyloids seeded with trace amounts of brain-extracted fibrils by 1 H-detected solid-state NMR at 80–90 kHz MAS frequency . The comparison with unseeded fibrils revealed marked structural differences as manifested by different chemical shifts and peak doubling, indicating two or more different structures in the brain-derived sample.…”

Amyloid fibril-UiO-66-NH2 aerogels for environmental remediation

“…Given the amorphous nature of the MOF/amyloid interface, high-resolution techniques such as gel-state NMR spectroscopy can be considered for future studies to provide additional insights regarding local structures and interactions. [25][26][27] The thermal stability of the aerogel was evaluated by thermogravimetric analysis (TGA) and shown in Fig. S5 (ESI †).…”