1H detection and dynamic nuclear polarization-enhanced NMR of Aβ1-42 fibrils

Abstract

Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments-namely, ¹H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on ¹³C,¹⁵N-enriched, and fully protonated samples of M₀Aβ_1-42 fibrils by high-field ¹H-detected NMR at 23.4 T and 18.8 T, and ¹³C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M₀Aβ_1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than ¹H detection. These results suggest that ¹H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

Keywords: 1H detection; amyloid β1-42; dynamic nuclear polarization; magic-angle spinning.

Fig. 1.

High-resolution ¹H-detected spectra recorded on fully protonated U-¹³C,¹⁵N-labeled M₀-Aβ_1-42 at 111.111 kHz MAS. (Top) 2D ¹³C-¹H CP-HSQC; (Bottom) 2D ¹⁵N-¹H CP-HSQC.

Fig. 2.

Representative restraints from the (A) 3D (H)NHH, (H)CHH and (B) (H)C(HH)CH spectra that show long-range ¹H-¹H contacts corresponding to the intramolecular structure of M₀-Aβ_1-42, highlighted in red. Asterisks mark diagonal peaks. (A) The NHH and CHH experiments establish contacts between F20H and I32γ₁₂ and A30α, between A30α and F20ζ F19β₃ and I42δ₁, and from F19α to I32γ₂/γ₁₂/δ₁. (B) The CCH experiments establish contacts between G33α and V36β and V36γ₁ and between I41β and G29α. (C) Display of intramolecular contacts found in the spectra shown in Fig. 3 on the lowest energy NMR structure of M₀Aβ_1-42 (Protein Data Bank ID 5kk3) (28). The contacts illustrated in this figure are listed in the legend for Fig. 3.

Fig. 3.

(A) DNP enhanced ¹³C spectra of 1,3-¹³C₂/2-¹³C/¹⁵N-labeled M₀Aβ_1-42 illustrating and enhancement of ε = 22 using (B) bis-nitroxide polarizing agent M-TinyPol. (C) DNP-enhanced ¹³C-¹³C CORD-RFDR spectra of 1,3-¹³C₂/2-¹³C/¹⁵N-labeled M₀Aβ_1-42. The resolution in the spectrum is comparable to that obtained at ambient temperatures, ∼0.6 ppm as indicated for I31Cβ-Cα. In red we show the long-range contacts that correspond to the intramolecular monomer structure of the fibril. (D) DNP-enhanced NCO and NCA (Right) spectra acquired using M-TinyPol as the polarizing agent. ω_r/2π = 40 kHz and T = 115 K.

Fig. 4.

Selected cross-sections from DNP-enhanced NCOCX (blue) and NCACX (red) spectra of 1,3-¹³C₂/2-¹³C/¹⁵N-labeled M₀-Aβ_1-42. Green circles indicate signals that reflect the interdimer contacts and the parallel-in-register arrangement of the fibrils. Asterisks mark diagonal peaks.