Amyloid fibril deposits observed in Alzheimer's disease comprise amyloid- (A) protein possessing a structured hydrophobic core and a disordered N-terminal domain (residues 1-16). The internal flexibility of the disordered domain is likely essential for A aggregation. Here, we used 2 H static solid-state NMR methods to probe the dynamics of selected side chains of the N-terminal domain of A 1-40 fibrils. Line shape and relaxation data suggested a two-state model in which the domain's free state undergoes a diffusive motion that is quenched in the bound state, likely because of transient interactions with the structured C-terminal domain. At 37 °C, we observed freezing of the dynamics progressively along the A sequence, with the fraction of the bound state increasing and the rate of diffusion decreasing. We also found that without solvation, the diffusive motion is quenched. The solvent acted as a plasticizer reminiscent of its role in the onset of global dynamics in globular proteins. As the temperature was lowered, the fraction of the bound state exhibited sigmoidal behavior. The midpoint of the freezing curve coincided with the bulk solvent freezing for the N-terminal residues and increased further along the sequence. Using 2 H R 1 measurements, we determined the conformational exchange rate constant between the free and bound states under physiological conditions. Zinc-induced aggregation leads to the enhancement of the dynamics, manifested by the faster conformational exchange, faster diffusion, and lower freezing-curve midpoints.
The structural polymorphism in β-amyloid (Aβ) plaques from Alzheimer disease (AD) has been recognized as an important pathological factor. Plaques from sporadic AD patients contain fibrillar deposits of various amyloid proteins/peptides, including posttranslational modified Aβ (PTM-Aβ) subtypes. Although many PTM-Aβs were shown to accelerate the fibrillation process, increase neuronal cytotoxicity of aggregates, or enhance the stability of fibrils, the contribution of PTM-Aβs to structural polymorphisms and their pathological roles remains unclear. We report here the NMR-based structure for the Ser-8-phosphorylated 40-residue Aβ (pS8-Aβ40) fibrils, which shows significant difference to the wild-type fibrils, with higher cross-seeding efficiency and thermodynamic stability. Given these physicochemical properties, the structures originated from pS8-Aβ40 fibrils may potentially dominate the polymorphisms in the mixture of wild-type and phosphorylated Aβ deposits. Our results imply that Aβ subtypes with “seeding-prone” properties may influence the polymorphisms of amyloid plaques through the cross-seeding process.
We investigate the variability in the dynamics of the disordered N-terminal domain of amyloid-b fibrils (Ab), comprising residues 1-16 of Ab 1-40 , due to post-translational modifications and mutations in the b-bend regions known to modulate aggregation properties. Using 2 H static solid-state NMR approaches, we compare the dynamics in the wild-type Ab fibrils in the threefold symmetric polymorph with the fibrils from three post-translational modification sequences: isoaspartate-D7, the phosphorylation of S8, and an N-terminal truncation DE3. Additional comparisons are made with the mutants in the b-bend region (residues 21-23) corresponding to the familial Osaka E22D deletion and D23N Iowa mutation. We also include the aggregates induced by Zn 2þ ions. The dynamics are probed at the F4 and G9 positions. The main motional model involves two free states undergoing diffusion and conformational exchanges with the bound state in which the diffusion is quenched because of transient interactions involving fibril core and other intrastrand contacts. The fraction of the bound state increases in a sigmoidal fashion with a decrease in temperature. There is clear variability in the dynamics: the phosphorylation of S8 variant is the most rigid at the G9 site in line with structural studies, the DE3 fibrils are more flexible at the G9 site in line with the morphological fragmentation pattern, the Zn-induced aggregates are the most mobile, and the two b-bend mutants have the strongest changes at the F4 site toward higher rigidity. Overall, the changes underlie the potential role of conformational ensembles in setting the stage for aggregation-prone states.
We employed deuterium solid‐state NMR techniques under static conditions to discern the details of the μs–ms timescale motions in the flexible N‐terminal subdomain of Aβ1–40 amyloid fibrils, which spans residues 1–16. In particular, we utilized a rotating frame (R1ρ) and the newly developed time domain quadrupolar Carr‐Purcell‐Meiboom‐Gill (QCPMG) relaxation measurements at the selectively deuterated side chains of A2, H6, and G9. The two experiments are complementary in terms of probing somewhat different timescales of motions, governed by the tensor parameters and the sampling window of the magnetization decay curves. The results indicated two mobile “free” states of the N‐terminal domain undergoing global diffusive motions, with isotropic diffusion coefficients of 0.7−1 ⋅ 108 and 0.3−3 ⋅ 106ad2 s−1. The free states are also involved in the conformational exchange with a single bound state, in which the diffusive motions are quenched, likely due to transient interactions with the structured hydrophobic core. The conformational exchange rate constants are 2−3 ⋅ 105 s−1 and 2−3 ⋅ 104 s−1 for the fast and slow diffusion free states, respectively.
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