Great Offset Difference Internuclear Selective Transfer

Abstract

Carbon-carbon dipolar recoupling sequences are frequently used building blocks in routine magic-angle spinning NMR experiments. While broadband homonuclear first-order dipolar recoupling sequences mainly excite intra-residue correlations, selective methods can detect inter-residue transfers and long-range correlations. Here, we present the great offset difference internuclear selective transfer (GODIST) pulse sequence optimized for selective carbonyl or aliphatic recoupling at fast magic-angle spinning, here, 55 kHz. We observe a 3- to 5-fold increase in intensities compared with broadband RFDR recoupling for perdeuterated microcrystalline SH3 and for the membrane protein influenza A M2 in lipid bilayers. In 3D (H)COCO(N)H and (H)CO(CO)NH spectra, inter-residue carbonyl-carbonyl correlations up to about 5 Å are observed in uniformly ¹³C-labeled proteins.

Figure 1

Simulations of carbonyl–carbonyl transfer using GODIST. (A) The GODIST pulse sequence element consisting of 32 2π pulses applied over 64 rotor periods (T_R) with the indicated phase cycle. (B) Schematic representation of the simulated four-spin system: two carbonyl carbons (C₁, C₂) and two aliphatic carbons (Cα, Cβ) with chemical shift anisotropy values (respectively, in kHz): [18; 16.5; 9.75; 9]. The C₁–C₂ distance and the C₂ chemical shift, δ2, were varied in the simulations of panels C–E and are shown in red. (C) Transfer as a function of the carbonyl offset difference (Δδ₁₂ = 180 – δ₂) for 2 to 4 Å distances, as indicated in the legend. (D,E) The total carbonyl signal (dashed lines) and the transferred signal (solid lines) as a function of mixing time for several distances. (D) Two-spin simulation with only carbonyl C₁ and C₂. (E) Four-spin simulation as shown in (B). All simulations used a 600 MHz proton Larmor frequency.

Figure 2

Comparison of RFDR (red) and GODIST (blue) transfers in 2D (H)CC spectra of perdeuterated microcrystalline SH3. The carbon carrier frequency was set to 50 ppm (A) and 173 ppm (B) for both sequences. For HC transfers, SPECIFIC–CP conditions^, were applied. For transfers from the aliphatic carbons (A) and from the carbonyl carbons (B), both GODIST and RFDR mixing was applied for 2.304 and 25.344 ms, respectively. For RFDR, 6 μs π pulses were applied. For GODIST, 36 μs 2π pulses were applied. All spectra were acquired at a 600 MHz spectrometer with 55.555 kHz MAS. XY-16 phase cycling^, was used for RFDR because it outperformed xy-8. Further experimental details are given in the Supporting Information (SI).

Figure 3

(A) ¹³C–¹³C projection of the 3D (H)COCO(N)H^GODIST spectrum of perdeuterated microcrystalline SH3 recorded with 25.344 ms of mixing. (B) Two strips extracted from the 3D (H)CO(CO)NH^GODIST spectrum at the nitrogen frequencies of L33 and V53. Distances indicated are taken from the crystal structure of SH3 (PDB code:2NUZ). Data was acquired at a 600 MHz spectrometer at 55.555 kHz MAS. The carbon carrier frequency was set to 185 ppm for the duration of mixing (further experimental details in the SI).

Figure 4

Six strips extracted from the 3D (H)CO(CO)NH^GODIST spectrum (9.216 ms mixing) at the amide nitrogen frequencies. The chemical shifts of nitrogen, carbon, and protons were taken from Movellan et al. The intensities of the correlated peaks are normalized with the peak intensities from the same experiment at zero mixing. Spectra were acquired at a 600 MHz spectrometer at 55.555 kHz MAS. The carbon carrier frequency was set to 185 ppm for the duration of mixing (further experimental details in the SI).