Multidimensional magic angle spinning NMR spectroscopy for site-resolved measurement of proton chemical shift anisotropy in biological solids

Abstract

The proton chemical shift (CS) tensor is a sensitive probe of structure and hydrogen bonding. Highly accurate quantum-chemical protocols exist for computation of (1)H magnetic shieldings in the various contexts, making proton chemical shifts potentially a powerful predictor of structural and electronic properties. However, (1)H CS tensors are not yet widely used in protein structure calculation due to scarcity of experimental data. While isotropic proton shifts can be readily measured in proteins even in the solid state, determination of the (1)H chemical shift anisotropy (CSA) tensors remains challenging, particularly in molecules containing multiple proton sites. We present a method for site-resolved measurement of amide proton CSAs in fully protonated solids under magic angle spinning. The approach consists of three concomitant 3D experiments yielding spectra determined by either mainly (1)H CSA, mainly (1)H–(15)N dipolar, or combined (1)H CSA and (1)H–(15)N dipolar interactions. The anisotropic interactions are recoupled using RN-sequences of appropriate symmetry, such as R12(1)(4), and (15)N/(13)C isotropic CS dimensions are introduced via a short selective (1)H–(15)N cross-polarization step. Accurate (1)H chemical shift tensor parameters are extracted by simultaneous fit of the lineshapes recorded in the three spectra. An application of this method is presented for an 89-residue protein, U-(13)C,(15)N-CAP-Gly domain of dynactin. The CSA parameters determined from the triple fits correlate with the hydrogen-bonding distances, and the trends are in excellent agreement with the prior solution NMR results. This approach is generally suited for recording proton CSA parameters in various biological and organic systems, including protein assemblies and nucleic acids.

Figure 1

Pulse sequences for (a) RN-¹H(¹⁵N_und) and RN-¹H(¹⁵N_dec), and (b) RN-¹⁵N 3D experiments. Rotor synchronized $R{12}_1^4$ symmetry rf pulses are applied during t₁ evolution time to reintroduce ¹H CSA or ¹H-¹⁵N dipolar interactions under MAS conditions. Empty and solid rectangles denote π/2 and π pulses, respectively. In (a) the time dependence of ¹H z-magnetization is monitored, either with application of the ¹⁵N decoupling π pulses (RN-¹H(¹⁵N_dec)) or without (RN-¹H(¹⁵N_und)). This optional decoupling pulse is colored in blue-grey. Site selective ¹H detection is accomplished by short-contact-time CP to ¹⁵N. In (b) the time evolution of ¹⁵N x-magnetization is monitored with refocusing of the chemical shift over a constant evolution period T. ¹⁵N-¹³C SPECIFIC-CP magnetization transfer is introduced following the t₂ evolution period, with the subsequent detection of the ¹³C signal during the t₃.

Figure 2

(a) Orientation of the CSA principal axes XYZ in the molecular frame of a hydrogen-bonded amide group. (b) Relative orientations of the NH bond vector and the CSA axes XYZ. (c) Relative orientations of the magnetic field B₀, the rotor axis, and the CSA principal axes XYZ of a single crystal, depicted at the moment in the rotor cycle at which the rotation angle is zero (see text); χ_m is the magic angle.

Figure 3

Simulated $R{12}_1^4$ spectra evaluated with D = 10 kHz and indicated principal components δ_XX – δ_iso and δ_ZZ – δ_iso of the ¹H CSA tensor (δ_YY – δ_iso is not indicated as it is equal to minus the sum of the other two components) and polar angles θ of the NH vector. The azimuthal angle φ is constant at 45°. Black: Calculated with average Hamiltonians. Blue: Calculated with full $R{12}_1^4$ Hamiltonian including three nearby protons. Other parameters: ¹H Larmor frequency 850 MHz, MAS frequency 14 kHz, 64 $R{12}_1^4$ time-domain increments of 1/14 ms for RN-¹H(¹⁵N_und) and RN-¹⁵N spectra and 2/14 ms for RN-¹H(¹⁵N_dec) spectra, 2/14 ms CP contact time, and 200 Hz Lorentzian line broadening. Number of crystallite orientations and γ angles are 320 and 4, respectively.

Figure 4

(a) The 2D NCA plane (at t₁ = 0) of the 3D R12₁^4-1H(¹⁵N_dec) spectrum of U-¹³C,¹⁵N-CAP-Gly domain of mammalian dynactin. The experiment was conducted at 19.9 T. Acquisition and processing parameters are presented in the Experiment section. (b) Experimental RN-¹H(¹⁵N_dec) lineshapes extracted from the 3D RN-¹H(¹⁵N_dec) spectrum for judiciously chosen CAP-Gly residues: H40 (terminus of β-sheet), Y46 (β-sheet), V47 (β-sheet), T50 (loop), T54 (loop), and C81 (loop).

Figure 5

Experimental (black) and best-fit simulated with full Hamiltonian (blue) $R{12}_1^4$ -symmetry based RN-¹H(¹⁵N_und), RN-¹H(¹⁵N_dec), and RN-¹⁵N lineshapes of select residues of U-¹³C,¹⁵N-CAP-Gly domain of mammalian dynactin.

Figure 6

Principal components of ¹H CSA tensors observed for 42 residues of U-¹³C,¹⁵N-CAP-Gly domain of mammalian dynactin, plotted as a function of the isotropic ¹H chemical shifts: δ_XX (circles), δ_YY (traingles), and δ_ZZ (diamonds). The straight lines represent the linear regression through similar CS plots of ubiquitin by Loth et al. (red: δ_XX = 2.4δ_iso − 5.2 ppm; δ_YY = 0.6δ_iso + 3.1 ppm; δ_ZZ = 2.1 ppm) and of GB3 by Yao et al. (black: δ_XX = 1.79δ_iso − 0.38 ppm; δ_YY = 1.22δ_iso − 2.28 ppm; δ_ZZ = −0.17δ_iso + 4.03 ppm). The three best fit lines through our experimental data are (not shown): δ_XX = 2.0δ_iso − 1.7 ppm (R_P = 0.85, p = 4.3×10⁻¹³); δ_YY = 2.0δ_iso − 8.3 ppm (R_P = 0.78, p = 7.5×10⁻¹⁰); δ_ZZ = −1.04δ_iso + 10.2 ppm (R_P = −0.65, p = 3.3×10⁻⁶).

Figure 7

Correlation between principal components of ¹H CSA tensors and hydrogen bond length in U-¹³C,¹⁵N-CAP-Gly domain of mammalian dynactin. (A) and (B) show the correlation between the span, δ_XX-δ_ZZ (black) and the H^⋯O and N^⋯O distance, respectively. In (C) and (D), correlations of the principal components δ_XX (red), δ_YY (grey), and δ_ZZ (blue) are shown with the H^⋯O and N^⋯O distance, respectively. The H^⋯O and N^⋯O distances are extracted from the MAS NMR structure of CAP-Gly (PDB code 2m02). The dashed lines in (A)–(D) correspond to equation (7). In (A), B = 10.9 ppm, C = 3.9 ppm Ų, and D = 1.0 Ų. The Pearson correlation coefficient R = 0.63, and the statistical significance p = 0.007. In (B), B = 11.5 ppm, C = 2.6 ppm Ų, and D = 2.0 Ų. The Pearson correlation coefficient R = 0.61, and the statistical significance p = 0.0046. The dotted lines in (C) and (D) represent the fits of the principal components of the CSA tensors to equation (7) with the fixed values of coefficient D (1.0 Ų and 2.0 Ų for H^⋯O and N^⋯O distances, respectively). In (C), the Pearson coefficient R and the statistical significance p are 0.48/0.05, 0.60/0.01, and −0.69/0.002 for δ_XX, δ_YY, and δ_ZZ, respectively. In (D), the corresponding parameters are 0.48/0.03, 0.51/0.02, and −0.60/0.005.