High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

Abstract

Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using δ subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences.

Fig. 1

Correlation of HA–CA (a), HB–CB (b), and N–CO (c) chemical shifts for the 81 a.a. unstructured part of the δ subunit of B. subtilis RNA polymerase. The ¹H and ¹³C chemical shifts depend mostly on particular amino acid residue, whereas ¹³CO and ¹⁵N frequencies are much better resolved and enable identification of backbone connectivity. (BMRB entry 16912)

Fig. 2

5D HACA(N)CONH technique. (a) Pulse sequence, ¹H, ¹³CA, and ¹⁵N evolution is in semi-constant-time mode: a_i = (t _i + Δ)/2, b _i = t _i(1−Δ/t _i^max)/2, c _i = Δ(1−t _i/t _i^max)/2 (where Δ stands for coherence transfer delays listed below, t _i is the evolution time in ith dimension, and t _i^max is maximal length of the evolution time delay). Delays were set as follows: Δ’_H-C = 2.6 ms, Δ_CA-N = 28.0 ms, Δ_CA-N-CO = 28.0 ms, Δ_N-CO = 28.0 ms, and Δ_N-H = 5.4 ms. The four-step phase cycle was used: ϕ₁ = x, −x, ϕ₂ = 2x, 2(−x) and Rec = ϕ₁ + ϕ₂. Simultaneous inversion of CA and CO spins was achieved using 6-element composite pulse (Shaka 1985). The coherence selection gradients (marked xyz) were applied at the magic angle. The phase ψ was inverted simultaneously with the last gradient pulse. (b) Coherence transfer in the peptide chain. H^N, N, and CO frequencies (filled rectangles) are ‘fixed’ for Fourier transform. Frames for HA and CA indicate the dimensions of 2D cross-sections obtained by SMFT procedure. (c) 2D spectral planes for the δ subunit of B. subtilis RNA polymerase, which were obtained by SMFT procedure performed on the 5D randomly sampled signal (Poisson disk sampling) with ‘fixed’ frequencies obtained from 3D HNCO peak list. Each cross-section contains two cross-peaks: for’fixed’ H_i^N, N_i and CO_i−1, the peaks correspond to HA_i–CA_i and HA_i−1–CA_i−1 correlations

Fig. 3

5D (HACA)CON(CA)CONH technique. (a) Pulse sequence, ¹⁵N (in t ₂ and t ₄) and ¹³CO (in t ₁ and t ₃) evolution is in semi-constant-time mode: a_i = (t _i + Δ)/2, b _i = t _i(1−Δ/t _i^max)/2, c _i = Δ(1−t _i/t _i^max)/2 (where Δ stands for coherence transfer delays listed below, t _i is the evolution time in ith dimension, and t _i^max is maximal length of the evolution time delay). Delays were set as follows: Δ_H-C = 3.7 ms, Δ’_H-C = 2.6 ms, Δ_CA-CO = 6.8 ms, Δ_CA-N = 28.0 ms, Δ_CA-N-CO = 28.0 ms, Δ_N-CO = 28.0 ms, Δ_N-H = 5.4 ms. The four-step phase cycle was used: ϕ₁ = x, −x, ϕ₂ = 2x, 2(−x) and Rec = ϕ₁ + ϕ₂. Simultaneous inversion of CA and CO spins was achieved using 6-element composite pulse (Shaka 1985). The coherence selection gradients (marked by xyz) were applied at the magic angle. The phase ψ was inverted simultaneously with the last gradient pulse. (b) Coherence transfer in the peptide chain. H^N, N, and CO frequencies (filled rectangles) are ‘fixed’ for Fourier transform. Frames for N and CO indicate the dimensions of 2D cross-sections obtained by SMFT procedure. (c) 2D spectral planes for the δ subunit of B. subtilis RNA polymerase, which were obtained by SMFT procedure performed on the 5D randomly sampled signal (Poisson disk sampling) with ‘fixed’ frequencies obtained from 3D HNCO peak list. Each cross-section contains two cross-peaks: for ‘fixed’ H_i^N, N_i and CO_i−1 the peaks correspond to N_i-CO_i−1 and N_i−1-CO_i−2 correlations

Fig. 4

5D HNCOCACB technique. (a) Pulse sequence, ¹³CO and ¹⁵N evolution is in semi-constant-time mode: a_i = (t _i + Δ)/2, b _i = t _i(1−Δ/t _i^max)/2, c _i = Δ(1−t _i/t _i^max)/2 (where Δ stands for listed below coherence transfer delays, t _i is the evolution time in ith dimension and t _i^max is maximal length of the evolution time delay). ¹³CA chemical shift evolution is in constant-time mode. Delays were set as follows: Δ_N-H = 5.4 ms, Δ_N-CO = 28.0 ms, Δ_CA-CO = 9.1 ms, and Δ_CACB = 14.3 ms. The four-step phase cycle was used: ϕ₁ = x, −x, ϕ₂ = 2x, 2(−x) and Rec = ϕ₁ + ϕ₂. The coherence selection gradients (marked by xyz) were applied at the magic angle. The phase ψ was inverted simultaneously with the last gradient pulse. (b) Coherence transfer in the peptide chain. H^N, N, and CO frequencies (filled rectangles) are ‘fixed’ for Fourier transform. Frames for CA and CB indicate the dimensions of 2D cross-sections obtained by SMFT procedure. (c) 2D spectral planes for the δ subunit of B. subtilis RNA polymerase, which were obtained by SMFT procedure performed on the 5D randomly sampled signal (Poisson disk sampling) with ‘fixed’ frequencies obtained from 3D HNCO peak list. Each cross-section contains one cross-peak: for ‘fixed’ H_i^N, N_i and CO_i−1 the peak corresponds to CA_i−1-CB_i−1 correlation

Fig. 5

5D (H)NCO(NCA)CONH technique. (a) Pulse sequence, ¹⁵N (in t ₁ and t ₄) and ¹³CO (in t ₃) evolution is in semi-constant-time mode: a_i = (t _i + Δ)/2, b _i = t _i(1−Δ/t _i^max)/2, c _i = Δ(1−t _i/t _i^max)/2 (where Δ stands for listed below coherence transfer delays, t _i is the evolution time in ith dimension and t _i^max is maximal length of the evolution time delay). Delays were set as follows: Δ_N-H = 5.4 ms, Δ_N-CO = 28.0 ms, Δ_CO–N-CA = 28.0 ms, Δ_N-CA = 28.6 ms, and Δ_CA-CO = 9.1 ms. The four-step phase cycle was used: ϕ₁ = x, −x, ϕ₂ = 2x, 2(−x) and Rec = ϕ₁ + ϕ₂. Simultaneous inversion of CA and CO spins was achieved using 6-element composite pulse (Shaka 1985). The coherence selection gradients (marked by xyz) were applied at the magic angle. The phase ψ was inverted simultaneously with the last gradient pulse. (b) Coherence transfer in the peptide chain. H^N, N, and CO frequencies (filled rectangles) are ‘fixed’ for Fourier transform. Frames for N and CO indicate the dimensions of 2D cross-sections obtained by SMFT procedure. (c) 2D spectral planes for the δ subunit of B. subtilis RNA polymerase, which were obtained by SMFT procedure performed on the 5D randomly sampled signal (Poisson disk sampling) with ‘fixed’ frequencies obtained from 3D HNCO peak list. Each cross-section contains two cross-peaks: for ‘fixed’ H_i^N, N_i and CO_i−1 the peaks correspond to N_i-CO_i−1 and N_i−1-CO_i−2 correlations

Fig. 6

6D (H)NCO(N)CACONH technique. (a) Pulse sequence, ¹⁵N (in t ₁ and t ₅) and ¹³CO (in t ₄) evolution is in semi-constant-time mode: a_i = (t _i + Δ)/2, b _i = t _i(1−Δ/t _i^max)/2, c _i = Δ(1−t _i/t _i^max)/2 (where Δ stands for listed below coherence transfer delays, t _i is the evolution time in ith dimension and t _i^max is maximal length of the evolution time delay). CA evolution (in t ₃) is in constant-time mode. Delays were set as follows: Δ_N-H = 5.4 ms, Δ_N-CO = 28.0 ms, Δ_CO-N-CA = 28.0 ms, Δ_N-CA = 28.6 ms, and Δ_CA-CO = 9.1 ms. The four-step phase cycle was used: ϕ₁ = x, −x, ϕ₂ = 2x, 2(−x) and Rec = ϕ₁ + ϕ₂. Simultaneous inversion of CA and CO spins was achieved using 6-element composite pulse (Shaka 1985). The coherence selection gradients (marked by xyz) were applied at the magic angle. The phase ψ was inverted simultaneously with the last gradient pulse. (b) Coherence transfer in the peptide chain. H^N, N, CO and CA frequencies (filled rectangles) are ‘fixed’ for Fourier transform. Frames for N and CO indicate the dimensions of 2D cross-sections obtained by SMFT procedure. (c) 2D spectral planes for the δ subunit of B. subtilis RNA polymerase, which were obtained by SMFT procedure performed on the 6D randomly sampled signal (Poisson disk sampling) with ‘fixed’ frequencies obtained from 4D HNCOCA peak list. Each plane contains two cross-peaks: for ‘fixed’ H_i^N, N_i, CO_i−1 and CA_i−1 the peaks correspond to N_i-CO_i−1 and N_i−1-CO_i−2 correlations

Fig. 7

Comparison of 2D N-CO cross-sections from 5D (H)NCO(NCA)CONH (a) and 6D (H)NCO(N)CACONH (b, c) experiments. In 5D experiment (a) the two pairs of correlation peaks overlap (E130/E131 with E170/E171, and E131/L132 with E171/I172) due to similarity of chemical shifts for all ‘fixed’ dimensions: H_L132^N and H_I172^N, N_L132 and N_I172, CO_E131 and CO_E171. The additional CA dimension in 6D experiment (b, c) enabled to differentiate and assign all peaks

Fig. 8

F ₁–F ₂ 2D cross-sections of HACA(N)CONH (a), HNCOCACB (b), and (H)NCO(NCA)CONH (c) 5D experiments, acquired for the δ subunit of B. subtilis RNA polymerase, show E168-E171correlations. The two intra- and inter-residual HA-CA correlation peaks are not resolved in (a); CA-CB correlation peaks in (b) have identical coordinates, however, the resolved pairs of N-CO peaks shown in (c) enable unambiguous sequential assignment