The process of transthyretin (TTR) misfolding and aggregation, including amyloid formation, appears to cause a number of degenerative diseases. During amyloid formation, the native protein undergoes a tetramer-to-folded monomer transition, followed by local unfolding of the monomer to an assembly-competent amyloidogenic intermediate. Here we use NMR relaxation dispersion to probe conformational exchange at physiological pH between native monomeric transthyretin (the F87M/L110M variant) and a small population of a transiently formed amyloidogenic intermediate. The dispersion experiments show that a majority of the residues in the β-sheet containing β-strands D, A, G and H undergo conformational fluctuations on μs-ms time scales. Exchange broadening is greatest for residues in the outer β-strand H, which hydrogen bonds to β-strand H’ of a neighboring subunit in the tetramer, but the associated structural fluctuations propagate across the entire β-sheet. Fluctuations in the other β-sheet are limited to the outer β-strand F, which packs against strand F’ in the tetramer, while the B, C, and E β-strands of this sheet remain stable. The structural changes were also investigated under more forcing amyloidogenic conditions (pH 6.4–3.7), where β-strand D and regions of the D-E and E-F loops were additionally destabilized, increasing the population of the amyloidogenic intermediate and accelerating amyloid formation. Strands B, C, and E appear to maintain native-like conformations in the partially unfolded, amyloidogenic state of wild type TTR. In the case of the protective mutant T119M, the conformational fluctuations are suppressed under both physiological and mildly acidic conditions, indicating that the dynamic properties of TTR correlate well with its aggregation propensity.

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F 19 → 23 Na triple quantum (TQ) cross polarization (TQCP) experiments and numerical simulations have been performed on the oxyfluoride NaMoO3F. Due to the orientation dependence of the quadrupolar tensor and thus the TQ nutation frequency (ωs,nut), only a fraction of the spins in the powder can match the Hartmann–Hahn condition at the same time, for a fixed F19 rf field strength (ω1I). Numerical simulations of the static TQCP process, for different single crystallite orientations, demonstrate that the most efficient TQCP occurs for parts of the powder where the quadrupolar splitting, Q′, is largest, and where the TQ coherences are pure eigenstates of the system, even though ωs,nut is smallest for these orientations. Under magic angle spinning (MAS) conditions, ωs,nut becomes time dependent and again, efficient TQCP is observed at times during the rotor period where Q′ is largest. TQCP intensities for different crystallites were calculated as a function of ω1I, for fixed Na23 rf field strengths, to obtain TQCP matching profiles and to determine optimum Hartmann–Hahn conditions. The TQCP matching profiles vary significantly with crystallite orientation and with spinning speed, due to the different time dependences of the quadrupolar interaction for the different orientations. Centerband matching conditions are observed at F19 rf fields, close to the minimum values of ωs,nut observed during the rotor period. Two different centerbands are observed for crystallite orientations where there are two different local minima for ωs,nut. The time dependence of ωs,nut results in higher-order (>2) sideband matching conditions. The experimentally observed static and MAS TQCP NMR matching profiles, and changes in the Na23 static and MAS second-order quadrupolar NMR lineshapes, could be rationalized on the basis of the calculated distributions of ωS,nut that occur within the powder. Optimum TQCP intensities were obtained with high Na23 rf power, to maximize ωs,nut, and slow MAS, to ensure efficient spin-locking; a further increase in intensity could be achieved by ramping the F19 rf field during the contact time. The efficiencies of the single quantum cross polarization (SQCP) multiple quantum (MQ) MAS and TQCP-MQMAS experiments were compared and were found to be very similar for NaMoO3F. Finally, the two-dimensional TQCP-MQMAS experiment was illustrated.

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Transition-metal ions (Cu2+ and Zn2+) play critical roles in the Abeta plaque formation. However, precise roles of the metal ions in the Abeta amyloidogenesis have been controversial. In this study, the molecular mechanism of the metal-induced Abeta oligomerization was investigated with extensive metal ion titration NMR experiments. Upon additions of the metal ions, the N-terminal region (1-16) of the Abeta (1-40) peptide was selectively perturbed. In particular, polar residues 4-8 and 13-15 were more strongly affected by the metal ions, suggesting that those regions may be the major binding sites of the metal ions. The NMR signal changes of the N-terminal region were dependent on the peptide concentrations (higher peptide concentrations resulted in stronger signal changes), suggesting that the metal ions facilitate the intermolecular contact between the Abeta peptides. The Abeta (1-40) peptides (>30 microM) were eventually oligomerized even at low temperature (3 degrees C), where the Abeta peptides are stable as monomeric forms without the metal ions. The real-time oligomerization process was monitored by 1H/15N HSQC NMR experiments, which provided the first residue-specific structural transition information. Hydrophobic residues 12-21 initially underwent conformational changes due to the intermolecular interactions. After the initial structural rearrangements, the C-terminal residues (32-40) readjusted their conformations presumably for effective oligomerization. Similar structural changes of the metal-free Abeta (1-40) peptides were also observed in the presence of the preformed oligomers, suggesting that the conformational transitions may be the general molecular mechanism of the Abeta (1-40) amyloidogenesis.

Elucidation of structural changes involved in protein misfolding and amyloid formation is crucial for unraveling the molecular basis of amyloid formation. Here we report structural analyses of the amyloidogenic intermediate and insoluble aggregates of transthyretin (TTR) using solution and solid-state NMR spectroscopy. Our solution NMR results show that one of the two main β-sheet structures (CBEF β-sheet) is maintained in the aggregation-competent intermediate, while the other DAGH β-sheet is more flexible on ms time scales. Magic-angle-spinning solid-state NMR revealed that AB loop regions interacting with strand A in DAGH β-sheet undergo conformational changes, leading to the destabilized DAGH β-sheet.

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