Broadband homonuclear correlation spectroscopy driven by combined R2(n)(v) sequences under fast magic angle spinning for NMR structural analysis of organic and biological solids

Abstract

We recently described a family of experiments for R2n(v) Driven Spin Diffusion (RDSD) spectroscopy suitable for homonuclear correlation experiments under fast MAS conditions [G. Hou, S. Yan, S.J. Sun, Y. Han, I.J. Byeon, J. Ahn, J. Concel, A. Samoson, A.M. Gronenborn, T. Polenova, Spin diffusion drive by R-symmetry sequencs: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids, J. Am. Chem. Soc. 133 (2011) 3943-3953]. In these RDSD experiments, since the broadened second-order rotational resonance conditions are dominated by the radio frequency field strength and the phase shifts, as well as the size of reintroduced dipolar couplings, the different R2n(v) sequences display unique polarization transfer behaviors and different recoupling frequency bandwidths. Herein, we present a series of modified R2n(v) sequences, dubbed COmbined R2n(v)-Driven (CORD), that yield broadband homonuclear dipolar recoupling and give rise to uniform distribution of cross peak intensities across the entire correlation spectrum. We report NMR experiments and numerical simulations demonstrating that these CORD spin diffusion sequences are suitable for broadband recoupling at a wide range of magnetic fields and MAS frequencies, including fast-MAS conditions (νr=40 kHz and above). Since these CORD sequences are largely insensitive to dipolar truncation, they are well suited for the determination of long-range distance constraints, which are indispensable for the structural characterization of a broad range of systems. Using U-(13)C,(15)N-alanine and U-(13)C,(15)N-histidine, we show that under fast-MAS conditions, the CORD sequences display polarization transfer efficiencies within broadband frequency regions that are generally higher than those offered by other existing spin diffusion pulse schemes. A 89-residue U-(13)C,(15)N-dynein light chain (LC8) protein has also been used to demonstrate that the CORD sequences exhibit uniformly high cross peak intensities across the entire chemical shift range.

Figure 1

(a) The general pulse sequence for 2D ¹³C-¹³C CORD homonuclear correlation experiments. An rf irradiation consisting of a series of composite pulses is applied on proton spins during the mixing time, τ_mix, with the irradiation sequences corresponding to: (b) basic CORD; (c) CORD_xix; (d) CORD_xy4. These irradiation schemes are composed of rotor-synchronized R2_n^v-type symmetry sequences: R2₁¹ (ν_1H = ν_r), R2₁² (ν_1H = ν_r), R2₂¹ (ν_1H = ν_r/2), R2₄² (ν_1H = ν_r/2).

Figure 2

Simulated dependence of the ¹³C-¹³C polarization transfer efficiency versus the isotropic chemical shift difference for R2₁¹ (basic PARIS) and R2₁¹ symmetry sequences for C₂H_n spin systems containing n = 1 proton (a, c) and n = 2 protons (b, d). The transfer efficiency is defined as 2I_2Z/(I_1Z + I_2Z), where I_1Z is the initial polarization on nucleus 1, and I_2Z is the polarization on spin 2 after the polarization transfer. The MAS frequency is ν_r = 40 kHz, and the mixing time is τ_mix = 150 ms. The ν_1H rf-field amplitude is equal to its nominal value of 20 (c,d) and 40 (a,b) kHz. The ¹³C-¹³C polarization transfer efficiency was simulated over the frequency difference of Δν_iso = ±50 kHz at the magnetic field of 14.1 T.

Figure 3

Simulated dependence of the ¹³C-¹³C polarization transfer efficiency versus the isotropic chemical shift difference for various composite rf pulse irradiations: (a) CORD, (b) CORD_xy4, (c) PARIS_xy, (d) SHANGHAI, (e) DREAM, and (f) fpRFDR. A C₂H₂ spin model (the same as in Figure 2b and 2d) was used for all the simulations. The MAS frequency is ν_r = 40 kHz, and the mixing time is τ_mix = 150 ms for (a-d), and 3 ms for (e) and (f). ¹³C-¹³C polarization transfer efficiency was simulated over the frequency difference of Δν_iso = ±50 kHz at the magnetic field of 14.1 T.

Figure 4

Simulated dependence of the ¹³C-¹³C polarization transfer efficiency as a function of the rf field strength, v_1H, and the resonant frequency difference, Δv_iso, for various composite rf pulse irradiations: (a) CORD, (b) CORD_xix, (c) CORD_xy4, (d) CORD_xy8, (e) PARIS_xy and (f) SHANGHAI. A C₂H₂ spin model was used for ¹³C-¹³C spin diffusion simulations at MAS frequency of ν_r = 40 kHz and magnetic field of 14.1 T, with a mixing time of τ_mix = 150 ms.

Figure 5

(a) 2D ¹³C-¹³C CORD correlation spectra recorded on U-¹³C,¹⁵N-alanine spun at 14.1 T, with the MAS frequency of ν_r = 40 kHz, the mixing period of τ_mix = 150 ms and the rf fields of ν_1H = 20 and 40 kHz. The first contour is set at 10×σ (σ is the noise rmsd) with the multiplier of 1.2. In (b), 1D traces extracted along the F₁ dimension of the 2D ¹³C-¹³C correlation spectra are shown, together with 1D traces extracted from homonuclear correlation spectra recorded by other composite pulse sequences: CORD (red), CORD_xix (blue), CORD_xy4 (black), PARIS_xy (green) and SHANGHAI (brown)

Figure 6

2D ¹³C-¹³C correlation NMR spectra of U-¹³C,¹⁵N-enriched histidine spun at the MAS frequency of ν_r = 40 kHz recorded at 14.1 T with CORD (a) and PARIS_xy (b) sequences, with the mixing time was τ_mix = 150 ms. The 1D traces extracted along the F₁ dimension are shown to illustrate the polarization transfer efficiency. The first contour in all spectra is set at 10×σ with the multiplier of 1.2.

Figure 7

1D traces along F₁ showing ¹³C-¹³C cross peaks extracted from 2D spin diffusion correlation NMR spectra of U-¹³C,¹⁵N-enriched histidine recorded at 14.1 T with various composite pulse sequences: CORD (red), CORD_xy4 (blue), PARIS_xy (purple), and SHANGHAI (black). The spinning frequency of ν_r = 40 kHz and the mixing time of τ_mix = 150 ms were used for all spin diffusion experiments. The diagonal auto-correlation peaks are not shown.

Figure 8

2D ¹³C-¹³C correlation NMR spectra of U-¹³C,¹⁵N-LC8 protein spun at the MAS frequency of ν_r = 40 kHz recorded at 14.1 T with CORD_xy4 (a), PARIS_xy (b) and SHANGHAI (c) sequences. The mixing time was τ_mix = 150 ms. The first contour in all spectra is set at 5×σ with the multiplier of 1.2.

Figure 9

2D CORD_xy4 ¹³C-¹³C correlation NMR spectra of U-¹³C,¹⁵N-LC8 protein spun at the MAS frequency of ν_r = 40 kHz and the magnetic field of 19.97 T, recorded with the mixing times of (a) 150 and (b) 500 ms. The first contour in all spectra is set at 5×σ with the multiplier of 1.2.

Figure 10

2D DREAM ¹³C-¹³C correlation NMR spectra of U-¹³C,¹⁵N-LC8 protein spun at the MAS frequency of ν_r = 40 kHz and the magnetic field of 19.97 T, recorded with the mixing times of (a) 4 and (b) 8 ms. High power ¹H decoupling with the rf field strength of 180 kHz was applied during DREAM mixing period. The first contour in all spectra is set at 5×σ with the multiplier of 1.2.

Figure 11

2D fpRFDR_xy16 ¹³C-¹³C correlation NMR spectra of U-¹³C,¹⁵N-LC8 protein spun at the MAS frequency of ν_r = 40 kHz and the magnetic field of 19.97 T, recorded with the mixing times of (a) 2.4 and (b) 9.6 ms. No ¹H decoupling was applied during the RFDR mixing period. The first contour in all spectra is set at 5×σ with the multiplier of 1.2.