MOMD Analysis of NMR Line Shapes from Aβ-Amyloid Fibrils: A New Tool for Characterizing Molecular Environments in Protein Aggregates

The microscopic-order-macroscopic-disorder (MOMD) approach for ²H NMR line shape analysis is applied to dry and hydrated 3-fold- and 2-fold-symmetric amyloid-Aβ₄₀ fibrils and protofibrils of the D23N mutant. The methyl moieties of L17, L34, V36 (C–CD₃), and M35 (S–CD₃) serve as probes. Experimental ²H spectra acquired previously in the 147–310 K range are used. MOMD describes local probe motion as axial diffusion (<i><b>R</b></i> tensor) in the presence of a potential, <i>u</i>, which represents the spatial restrictions exerted by the molecular surroundings. We find that <i>R</i>_∥ = (0.2–3.3) × 10⁴ s^–1, <i>R</i>_⊥ = (2.2–2.5) × 10² s^–1, and <b><i>R</i></b> is tilted from the ²H quadrupolar tensor at 60–75°. The strength of <i>u</i> is in the (2.0–2.4) <i>kT</i> range; its rhombicity is substantial. The only methyl moieties affected by fibril hydration are those of M35, located at fibril interfaces. The associated local potentials change form abruptly around 260 K, where massive water freezing occurs. An independent study revealed unfrozen “tightly-peptide-bound” water residing at the interfaces of the 3-fold-symmetric Aβ₄₀ fibrils and at the interfaces of the E22G and E22Δ Aβ₄₀-mutant fibrils. Considering this to be the case in general for Aβ₄₀-related fibrils, the following emerges. The impact of water freezing is transmitted selectively to the fibril structure through interactions with tightly-peptide-bound water, in this case of M35 methyl moieties. The proof that such waters reside at the interfaces of the 2-fold-symmetric fibril, and the protofibril of the D23N mutant, is new. MOMD provides information on the surroundings of the NMR probe directly via the potential, <i>u</i>, which is inherent to the model; a prior interpretation of the same experimental data does so partially and indirectly (see below). Thus, MOMD analysis of NMR line shapes as applied to amyloid fibrils/protein aggregates emerges as a consistent new tool for elucidating the properties of, and processes associated with, molecular environments in the fibril