Facile measurement of ¹H-¹5N residual dipolar couplings in larger perdeuterated proteins

Abstract

We present a simple method, ARTSY, for extracting ¹J(NH) couplings and ¹H-¹⁵N RDCs from an interleaved set of two-dimensional ¹H-¹⁵N TROSY-HSQC spectra, based on the principle of quantitative J correlation. The primary advantage of the ARTSY method over other methods is the ability to measure couplings without scaling peak positions or altering the narrow line widths characteristic of TROSY spectra. Accuracy of the method is demonstrated for the model system GB3. Application to the catalytic core domain of HIV integrase, a 36 kDa homodimer with unfavorable spectral characteristics, demonstrates its practical utility. Precision of the RDC measurement is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC spectrum, and is approximately given by 30/(S/N) Hz.

Figure 1

The ARTSY pulse scheme used to measure ¹H-¹⁵N RDCs. Narrow and wide pulses correspond to 90° and 180° flip angles, respectively. Unless marked, RF phases of all pulses are x. The ¹H carrier frequency is set to the water frequency, and ¹⁵N, ¹³C′, and ¹³C^α carriers are set to 117, 176, and 56 ppm, respectively. Open ¹H pulses are 90°, with a shape corresponding to the center lobe of a sinc(x) function. The last non-selective 180° ¹H pulse is flanked by two low power 1.2-ms rectangular pulses (90° each) which aid in H₂O signal suppression (Piotto et al. 1992). The 180° proton pulse during this element is offset slightly (by ~150 μs) relative to the 180° ¹⁵N pulse to allow inclusion of the g₇ decoding gradient during the second τ delay without requiring first order phase correction in the ¹H dimension. The open composite ¹⁵N pulse is of the 90_x-210_y-90_x type, compensating for both offset and RF inhomogeneity (Freeman et al. 1980), and is applied at time point d (reference) or c (attenuated experiment). The delay T is set to ~1/J_NH or 10.75 ms. For deriving the coupling values, using eqs 2 or 3, dephasing during the pulses needs to be taken into account, and a value T+ε is used in eqs 2 and 3, with ε = (4/π)H₉₀−κ, where H₉₀ equals the ¹H 90° pulse width and κ equals the total duration of the ¹⁵N composite pulse. Other delays: δ = δ′ = ~1/4J_NH = 2.71 ms; Δ = 0.75 ms; τ = 1.66 ms. The ¹³C′ and ¹³C^α decoupling pulses (only needed for proteins also enriched in ¹³C) are applied with a RF field strength of $\mathrm{\Delta }f/\sqrt{3}$, where Δf is the difference in Hz between the centers of the ¹³C′ and ¹³C^α chemical shift regions. Gradients: g₂ = 0.7 G/cm; g_1,3,4,5,6,7 are sine-bell shaped with durations of 1.0, 0.5, 0.5, 1.0, 0.8, and 0.1013 ms, respectively, and strengths of 20.3, 8.4, ± 42, 2.3, 4.9, and 42 G/cm at their mid-points. Phase cycling: φ₁ = x, −x; φ₂ = 2(y), 2(−y); φ₃ = −y; φ₄ = 4(x), 4(−x);φ₅ = 4(−y), 4(y); φ_rec = y, 2(−y), y, −y, 2(y), −y. Phase φ₅ and the duration of δ′ may be adjusted to reduce anti-TROSY artifacts according to the Clean TROSY method. Given the average relative intensity of the anti-TROSY artifacts, φ₅ is generally decreased and δ′ is shortened according to eq 10 of (Schulte-Herbruggen and Sorensen 2000). In practice, anti-TROSY artifacts were vanishingly weak in our integrase spectra, and therefore no such compensation was used. Quadrature in the ¹⁵N dimension uses the regular gradient coherence selection method, inverting the phases of φ₃, φ₅ and the sign of g₄ for the second FID collected for each value of t₁ (Kay et al. 1992; Pervushin et al. 1998).

Figure 2

Spectral data used for measuring ¹H-¹⁵N RDCs in IN^50-212. Small regions of the reference (a) and attenuated (b) ¹⁵N-¹H TROSY spectra, recorded at 900 MHz in the presence of 4% (w/v) C12E5 PEG/hexanol (Ruckert and Otting 2000). Peak positions are determined using the reference spectra and then mapped directly on to the attenuated spectra. Positive and negative intensity contours are red and blue, respectively. In the attenuated spectra, negative (positive) peaks correspond to resonances with |J+D| greater than (less than) 93 Hz.

Figure 3

Best fit correlation between ARTSY-derived ¹D_NH RDCs for homodimeric IN^50-192 and the coordinates of PDB entry 1BIS, chain B (Goldgur et al. 1998). The alignment tensor was best-fitted using the DC program in the NMRPipe software package (Delaglio et al. 1995). Black symbols correspond to the 97 RDCs used in fitting (Q = 0.279). Green squares and orange triangles correspond to the active site loop (residues 140–152) and the loop between helices α₅ and α₆ (residues 187–194) (see colored regions on the inset structure) and were not included in the RDC fit. These amides were shown to be dynamic in ¹⁵N relaxation measurements (Fitzkee et al. 2010). Slightly better fits are obtained using the chains of PDB entry 1BIU (Q ~ 0.26), but its chains lack density for residues 141–148 of the catalytic loop (Goldgur et al. 1998).