Homonuclear decoupling for enhancing resolution and sensitivity in NOE and RDC measurements of peptides and proteins

Abstract

Application of band-selective homonuclear (BASH) (1)H decoupling pulses during acquisition of the (1)H free induction decay is shown to be an efficient procedure for removal of scalar and residual dipolar couplings between amide and aliphatic protons. BASH decoupling can be applied in both dimensions of a homonuclear 2D NMR experiment and is particularly useful for enhancing spectral resolution in the H(N)-H(α) region of NOESY spectra of peptides and proteins, which contain important information on the backbone torsion angles. The method then also prevents generation of zero quantum and Hz(N)-Hz(α) terms, thereby facilitating analysis of intraresidue interactions. Application to the NOESY spectrum of a hexapeptide fragment of the intrinsically disordered protein α-synuclein highlights the considerable diffusion anisotropy present in linear peptides. Removal of residual dipolar couplings between H(N) and aliphatic protons in weakly aligned proteins increases resolution in the (1)H-(15)N HSQC region of the spectrum and allows measurement of RDCs in samples that are relatively strongly aligned. The approach is demonstrated for measurement of RDCs in protonated (15)N/(13)C-enriched ubiquitin, aligned in Pf1, yielding improved fitting to the ubiquitin structure.

Keywords: Diffusion anisotropy; IDP; Liquid crystal; NOESY; RDC; Residual dipolar coupling; Synuclein; Ubiquitin; Weak alignment.

Fig. 1

Pulse sequences using band-selective homonuclear decoupling during data acquisition. (A) Pulse scheme for recording a 1D spectrum of the amide region of peptides and proteins, with BASH decoupling of aliphatic protons. (B) 2D NOESY pulse scheme, with BASH decoupling during both t₁ evolution and t₂ detection periods. The BASH decoupling during t₁ evolution only requires a single pulse pair, with the band-selective pulse applied to the H^α region, and decouples it from ¹H^β and ¹H^N protons. Decoupling in this dimension will not remove geminal ¹H^α2- ¹H^α3 J couplings in Gly, nor ¹H^α- ¹H^β J couplings in Thr and Ser residues, as these typically resonate in the same spectral region as the ¹H^α protons selected by the BASH pulse. (C) Pulse scheme for the ¹H-¹⁵N 2D HSQC spectrum with BASH decoupling during t₂. Shaped pulses marked r are REBURP 180° pulses [23] (1.6 ms centered at 9.67 ppm for the HSQC on Pf1-aligned ubiquitin at 500 MHz and 2 ms centered at 8.6 ppm for the 1D and 2D NOESY on the hexapeptide at 747 MHz). The shaped pulse marked s is a 1-ms (at 747 MHz) sine bell shaped selective ¹H^α pulse, centered at 4.2 ppm. The shaped pulse marked e is an EBURP1 pulse [23], centered at 8.6 ppm, of duration 2.0 ms (747 MHz). H₂O presaturation is used (25 Hz RF field strength) during the delay between scans. Specific parameters for scheme (A): t/4n = 12 ms; n = 9; phase cycling: ϕ₁ = x, x, −x, −x; ϕ₂ = −x, x; ϕ₃ = x, −x; ϕ₄ = −x and ϕ₅ = x for the odd blocks, and inverted for even numbered values of the loop counter, n; Rec. = x, −x; delays: τ= 83 μs; δ = 2.2 ms; Gradient durations: G_1,2,3,4,5,6 = 1.2, 2, 0.3, 0.37, 0.3, 0.37 ms; Gradients are all sine bell shaped with maximum amplitude (x,y,z) G_1,2,3,4,5,6 = (25.2, 25.2, 35.7), (0, 0, 38.5), (18.3, 0, 0), (0, 18.3, 0), (16.3, 11.4, 1.4), (16.3, 1.5, 15.4) G/cm; Specific parameters for scheme (B): t₂/4n = 12 ms; n = 6; phase cycling: ϕ₁ = 8(x),8(−x) ϕ₂ = x, x, −x, −x; ϕ₃ = 4(x),4(−x); ϕ₄ = −x, x; ϕ₅ = −x and ϕ₆ = x for the odd-numbered values of the loop counter, n, and inverted for even values of n; Rec. = x, −x, −x, x; delay: δ = 2.2 ms; Gradient durations: G_{1,2,3, 6,7,8,9,10,11} = 1.2, 0.37, 0.3, 5, 2, 0.3, 0.37, 0.3, 0.37 ms; Gradients 1–3 and 6–11 are all sine bell shaped with maximum amplitude (x,y,z) G_{1,2,3,6,7,8,9,10,11} = (25.2, 25.2, 35.7), (18.3, 0, 0), (16.3, 11.4, 1.4), (15.3, 1.5, 15.4), (0, 0, 38.5), (18.3, 0, 0), (0, 18.3, 0), (16.3, 11.4, 1.4), (15.3, 1.5, 15.4) G/cm; gradients G4 and G5 are rectangular with amplitudes G_4,5= (0.5, 0.5, 0.7) and (-0.6, -0.6, -0.9) G/cm and durations slightly less than half the NOE mixing time, τ_noe/2. The open pulse in the center of τ_noe represents a 90_x210_y90_x composite inversion pulse. Quadrature in the t₁ dimension is accomplished by State-TPPI phase incrementation of ϕ₂. Specific parameters for scheme (C): t₂/4n = 5 ms; n=4; phase cycling: ϕ₁ = x, −x; ϕ₂ = x, x, −x, −x; ϕ₃ = x and ϕ₄ = −x for the odd values of n and inverted for even values; Rec. = x, −x, −x, x; delays: δ = 1.9 ms; Gradient durations: G_{1,2,3,4,5,6,7,8,9} = 1.7, 1.7, 1.1, 1.2, 1.4, 0.21, 0.21, 0.21, 0.21 ms; Gradients are all sine bell shaped with maximum amplitude G_{1,2,3,4,5,6,7,8,9} = 35.7, 2.1, 28.7, 20.8, 32.9, 14.7, 21, 25.9, 32.9 G/cm; Quadrature in the t₁ dimension is accomplished by State-TPPI phase incrementation of ϕ₁. The pulse sequences and parameters for all three pulse schemes can be downloaded from http://spin.niddk.nih.gov/bax/pp/

Fig. 2

Comparison of BASH-decoupled and regular ¹H spectra of a linear hexapeptide, Ac-VAAAEK-NH₂ (10 mM), recorded at 747 MHz ¹H frequency, 25 °C, pH 6.0, 20 mM sodium phosphate, with the pulse schemes of Fig.1A and 1B. The peptide corresponds to residues 16–21 of α-synuclein. (A) Amide region of the regular ¹H spectrum, recorded with weak (25 Hz RF field) presaturation of the H²O resonance. (B) BASH decoupled amide region. (C) H^α-H^N region of the regular 2D NOESY spectrum, recorded with a 200 ms NOE mixing time, using acquisition times of 133 ms (t₁) and 274 ms (t₂). NOEs for this peptide are very weak, due to the condition ωτ_c ≈ 1, and cross peaks are dominated by coherent (zero quantum) contributions. The only NOE cross peak clearly visible is the sequential NOE from A2-H^α to A3-H^N and corresponds to a cross relaxation rate of −0.01±0.002 s⁻¹. Blue contours correspond to positive intensity (with the diagonal being positive); red depicts negative intensity. (D) H^α-H^N region of the 2D BASH-NOESY spectrum, recorded with the pulse scheme of Fig. 1B, using a 200 ms NOE mixing time and acquisition times of 319 ms (t₁) and 270 ms (t₂). Blue cross peaks correspond to ωτ_c> 1.1 and all are sequential H^α-H^N NOEs; red cross peaks have negative intensity, i.e. ωτ_c < 1.1, and with one exception (V1-A2) are all intraresidue and labeled by residue type and number. The absolute value of the intraresidue H^α-H^N NOE of E5 falls below the detection threshold (σ < 10⁻³ s⁻¹).

Fig. 3

Comparison of a small region of the BASH-decoupled and regular ¹H-¹⁵N HSQC spectrum of human ubiquitin (400 μM) in a liquid crystalline Pf1 suspension (13 mg/mL; 140 mM NaCl; pH 6.0, 25 °C), recorded at 500 MHz with (A) the regular HSQC experiment, and (B) the same pulse scheme, but using BASH decoupling during t₂ acquisition (Fig. 1B).

Fig. 4

Comparison of the average signal to noise ratio observed for the ¹³C^α-¹H^α doublet components in a ¹H^α-coupled 3D HN(CO)CA experiment recorded with and without BASH decoupling during data acquisition on the same sample used for Fig. 3. The spectrum without BASH decoupling was recorded without water presaturation, the BASH-HN(CO)CA spectrum was recorded with weak water presaturation, causing some of the signals to be weaker in the BASH decoupled spectrum. The actual RDCs extracted from these two spectra are listed in Supplementary Material Table S1.