TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins

Abstract

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.

Figure 1

Experimental scheme for the [¹⁵N, ¹H]-TROSY-HNCA experiment. The radio-frequency pulses on ¹H, ¹⁵N, ¹³C^α, ¹³CO, ²H, and ¹H^α are applied at 4.7, 118, 56, 177, 3.6, and 4.7 ppm, respectively. Narrow and wide black bars indicate nonselective 90° and 180° pulses. Sine bell shapes on the lines marked ¹H and ¹H^α indicate selective 90° pulses. On the line marked ¹³CO, three selective 180° pulses are applied off-resonance with a duration of 120 μs and a Gaussian shape. The line marked PFG indicates the durations and amplitudes of pulsed magnetic field gradients applied along the z-axis: G₁: 800 μs, 15 G/cm; G₂: 800 μs, 9 G/cm; and G₃: 800 μs, 22 G/cm. The delays are τ = 2.7 ms and T = 44 ms. The phase cycle is: φ₁ = {y, −y, −x, x}; φ₂ = {4x, 4(−x)}; φ₃ = {−y}; φ₄ = {−y}; φ_rec = {y, −y, −x, x, −y, y, x, −x}, with all other radio-frequency pulses applied with phase x. A phase-sensitive spectrum in the ¹⁵N(t₁) dimension is obtained by recording a second FID for each t₁ value, with φ₁ = {y, −y, x, −x}, φ₃ = {y} and φ₄ = {y}, and data processing as described by Kay et al. (41). Quadrature detection in the ¹³C^α(t₂) dimension is achieved by the States-TPPI method (42) applied to the phase φ₂. The use of water flip-back pulses (43) at times a and e ensures that the water magnetization stays aligned along +z throughout both the ct period T and the data acquisition period t₃. Residual transverse water magnetization is suppressed immediately before data acquisition (44). The scheme is used for two alternative experiments. For ²H-labeled proteins, ²H-decoupling during t₂ is achieved with WALTZ-16 composite pulse decoupling (45) at a field strength of γB₂ = 2.5 kHz. For measurements with protonated proteins, selective ¹H^α-decoupling during the ¹³C^α(t₂) evolution period is applied instead, using a DIPSI-2 decoupling scheme (46) with γB₂ = 0.51 kHz. The two selective pulses on the water resonance before and after DIPSI-2 ensure the correct treatment of the water during the subsequent t₁ and t₂ evolution periods.

Figure 2

Comparison of a [¹⁵N, ¹H]-TROSY-HNCA recorded with the scheme of Fig. 1 and b conventional HNCA (13) using ¹H DIPSI-2 decoupling with γB₂ = 3.13 kHz during t₁ and t₂, and ¹⁵N WALTZ-16 decoupling with γB₂ = 1.6 kHz during acquisition. Both experiments were recorded with a 1 mM solution of uniformly ²H/¹³C/¹⁵N-labeled gyrase-23B. 26(t₁) × 32(t₂) × 512(t₃) complex points were accumulated, with t_1max(¹⁵N) = 21.7, t_2max(¹³C^α) = 6.4, and t_3max(¹H) = 48.7 ms. Fifty-six scans per increment were acquired, resulting in a total measuring time of 38 h per 3D spectrum. Corresponding [ω₂(¹³C),ω₃(¹H)] strips from the two 3D experiments centered about the ¹H^N chemical shifts were taken at the ¹⁵N chemical shifts of residues 47–50. Because no decoupling during t₁ and t₃ is used in TROSY, the amide ¹H^N and ¹⁵N resonances in a are shifted in both dimensions by ≈45 Hz relative to the corresponding resonances in b. The sequence-specific assignments are indicated at the top by the one-letter amino acid symbol and the residue number in the amino acid sequence. In both spectra, dashed lines indicate sequential connectivities that could be reliably identified (see text). (a′ and b′) cross sections along the ω₂(¹³C) dimension through the four [ω₂(¹³C),ω₃(¹H)] strips at the ω₃(¹H) positions indicated at the bottom in a and b, respectively, where the complete chemical shift range acquired in the ω₂(¹³C) dimension is plotted.

Figure 3

Plots of the relative signal intensities, I_rel, along the amino acid sequence of uniformly ²H/¹³C/¹⁵N-labeled gyrase-23B (1 mM protein concentration in 95% H₂O/5% D₂O at pH 6.5 and T = 20°C). (a) Sequential correlation peaks (ω₂(¹³C_i−1^α)/ω₃(¹H_i^N)) in [¹⁵N, ¹H]-TROSY-HNCA. (b) Same as a measured with conventional HNCA (13). (a′) Intraresidual correlation peaks (ω₂(¹³C_i^α)/ω₃(¹H_i^N)) in [¹⁵N, ¹H]-TROSY-HNCA. (b′) Same as a′ measured with conventional HNCA. Both spectra were recorded with the same experimental conditions and processed identically, as described in the text. The α-helical and β-sheet regions in the x-ray structure of gyrase-23B (22) are identified in b′ by open and filled bars, respectively.

Figure 4

(a) Experimental scheme for the [¹⁵N, ¹H]-TROSY-HNCO experiment. All experimental parameters and the phase cycle are the same as in the [¹⁵N, ¹H]-TROSY-HNCA scheme of Fig. 1. (b and c) Show, respectively, cross sections along the ω₃(¹H) dimension through four peaks of the 3D [¹⁵N, ¹H]-TROSY-HNCO spectrum and the conventional 3D HNCO spectrum (30) of uniformly ²H/¹³C/¹⁵N-labeled gyrase-23B (1 mM protein concentration in 95% H₂O/5% D₂O at pH 6.5 and T = 20°C). 26(t₁) × 30(t₂) × 512(t₃) complex points were accumulated, with t_1max (¹⁵N) = 10.8, t_2max(¹³CO) = 12.0, and t_3max(¹H) = 48.7 ms. Eight scans per increment were acquired, resulting in a total measuring time of 7 h per 3D spectrum. At the top of each panel, the sequence-specific assignment is indicated by the one-letter amino acid symbol and the residue number in the amino acid sequence, and the ω₁(¹⁵N) and ω₂(¹³CO) chemical shifts are indicated in parentheses.

Figure 5

Relative signal amplitudes, A, calculated during the ct ¹⁵N evolution and ¹H acquisition periods for [¹⁵N, ¹H]-TROSY-HNCA (thick lines) and conventional HNCA (thin lines) at a magnetic field strength of 750 MHz for variable isotropic rotational correlation times, τ_c, in the range from 5 to 80 ns, which corresponds to protein sizes in H₂O solution at 20°C from ≈7 kDa at τ_c = 5 ns to ≈260 kDa at τ_c = 80 ns. The solid and dashed lines represent the signal amplitudes, A, for a ¹⁵N–¹H^N moiety in a β-sheet of a deuterated and a protonated ¹³C/¹⁵N-labeled protein, respectively (see Table 1 for the parameters used). (Inset) An expanded plot for the range of A from 0 to 1. The dotted vertical line indicates the τ_c value of 15 ns estimated for the 23-kDa proteins gyrase-23B and FimC.