3D MAS NMR Experiment Utilizing Through-Space 15N-15N Correlations

Abstract

We demonstrate a novel 3D NNC magic angle spinning NMR experiment that generates ¹⁵N-¹⁵N internuclear contacts in protein systems using an optimized ¹⁵N-¹⁵N proton assisted recoupling (PAR) mixing period and a ¹³C dimension for improved resolution. The optimized PAR condition permits the acquisition of high signal-to-noise 3D data that enables backbone chemical shift assignments using a strategy that is complementary to current schemes. The spectra can also provide distance constraints. The utility of the experiment is demonstrated on an M₀Aβ_1-42 fibril sample that yields high-quality data that is readily assigned and interpreted. The 3D NNC experiment therefore provides a powerful platform for solid-state protein studies and is broadly applicable to a variety of systems and experimental conditions.

Figure 1:

The 3D NNC pulse sequence is shown in A) with phase cycling: ϕ₁ = 02, ϕ₂ = 0022, ϕ₃ = 1, ϕ₄ = 2002, ϕ₅ = 0123, ϕ₆ = 2002 0220, ϕ_rec = 0123 2301. The CP, PAR, and SPECIFIC-CP blocks are color coded to arrows in (B) illustrating the corresponding magnetization transfer. Additionally, B) contrasts the mode of mixing for the NNC experiment, and commonly used protocols (NCACX, NCOCX, CONCA), illustrating their complementary information due to reliance on separate contacts. C) visualizes the internuclear distances and corresponding dipole coupling values for a range of ¹⁵N-¹⁵N contacts in α-helices and parallel and anti-parallel β-sheets. The residue interval label (i.e. i ± 1, i ± 2, etc.) is color coded to the lines/arrows illustrating transfer. The ¹⁵N-¹⁵N inter-strand β-sheet distances are given as approximate averages.

Figure 2:

2D ¹⁵N-¹⁵N homonuclear correlation spectra on M₀Aβ_1–42. A) gives a comparison of 1D traces extracted from the 2D spectra in B)-D) as indicated by a gray line, with a comparison of measured signal-to-noise (S/N) and total experiment times. Panels B)-D) show a τ_mix = 12.8 ms ¹⁵N-¹⁵N RFDR (green), a τ_mix = 20 ms ¹⁵N-¹⁵N PAR (red), and a τ_mix = 4 s ¹⁵N-¹⁵N PDSD (blue), respectively. Cross-peak assignments are shown.

Figure 3:

3D NNC^α projections of M₀Aβ_1–42. One CN projection (CN₁) shows one peak per residue correlating the nitrogen and the C^α of the same amino acid i. The second CN projection (CN₂) is identical to CN₁ plus additional peaks due to NN-mixing. For each C^α/N pair (which forms the diagonal peak) there are peaks that correspond to nearby ¹⁵N sites, most prominently the i±1 backbone amide ¹⁵N nuclei. The third projection (NN) is identical to a 2D ¹⁵N-¹⁵N PAR spectrum showing the backbone nitrogen of residue i as diagonal and other peaks (mostly backbone nitrogen of residues i±1) as cross-peaks. The 3D spectrum was recorded on a Bruker Avance III 800 MHz spectrometer at ω_r/2π = 20 kHz with temperature set to 277 K. A more detailed assignment is shown in Figure S1.

Figure 4:

Strip plot of the 3D NNC^α of M₀Aβ_1–42. Each strip displays the diagonal peak of residue i (blue) and several cross-peaks (red). The most prominent cross-peaks at 20 ms PAR mixing are from residues i±1, although cross-peaks from residues i±2 can be present (*). The sequential backbone walk is indicated by dashed lines. Colors do not indicate sign and are solely used as visual aids.