Water irradiation devoid pulses enhance the sensitivity of 1H,1H nuclear Overhauser effects

Abstract

The nuclear Overhauser effect (NOE) is one of NMR spectroscopy's most important and versatile parameters. NOE is routinely utilized to determine the structures of medium-to-large size biomolecules and characterize protein-protein, protein-RNA, protein-DNA, and protein-ligand interactions in aqueous solutions. Typical [¹H,¹H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates ¹H-detected spectra and minimize NOE intensity losses due to unwanted polarization exchange between water and labile protons. However, at high- and ultra-high magnetic fields, the excitation of the water signal during the execution of the NOESY pulse sequences may cause significant attenuation of NOE cross-peak intensities. Using an evolutionary algorithm coupled with artificial intelligence, we recently designed high-fidelity pulses [Water irrAdiation DEvoid (WADE) pulses] that elude water excitation and irradiate broader bandwidths relative to commonly used pulses. Here, we demonstrate that WADE pulses, implemented into the 2D [¹H,¹H] NOESY experiments, increase the intensity of the NOE cross-peaks for labile and, to a lesser extent, non-exchangeable protons. We applied the new 2D [¹H,¹H] WADE-NOESY pulse sequence to two well-folded, medium-size proteins, i.e., the K48C mutant of ubiquitin and the Raf kinase inhibitor protein. We observed a net increase of the NOE intensities varying from 30 to 170% compared to the commonly used NOESY experiments. The new WADE pulses can be easily engineered into 2D and 3D homo- and hetero-nuclear NOESY pulse sequences to boost their sensitivity.

Keywords: Artificial intelligence; Evolutionary algorithm; GENETICS-AI; Nuclear Overhauser effect; Pulse design; WADE pulses; [1H,1H] NOESY.

Figure 1.

Water suppression pulse schemes utilized for the 2D [¹H, ¹H] NOESY experiments. (A) The 3–9-19 sequence with total durations of 3.02 ms and 2.4ms for interpulse delays of 200 μs and 75 μs, respectively. (B) The WATERGATE sequence with a total duration of 6.04 ms using a selective pulse of 2 ms. (C) The WATERGATE sequence with water flip-back pulses for a total duration of 8.03 ms. (D) The excitation sculpting sequence with a total duration of 8.04ms. (E) The WADE sequence with a maximum RF amplitude of 5 kHz and a total duration of 5.01 ms. Unfilled wide and narrow rectangular shapes represent hard π and π /2 pulses, respectively. Unfilled shapes in ¹H channel represent water-selective π/2 or π pulses. Filled shapes represent gradient pulses of 1 ms duration.

Figure 2.

Contour plots showing the responses of the transverse magnetization components (M_x or M_y) to the water suppression schemes showed in Figure 1 as a function of RF amplitude (B₁), and for a bandwidth of 25 kHz. (A) Response of the M_x component to the Grad-3919-Grad sequence with an interpulse delay of 200 μs. The total length for the suppression sequence was 3.02 ms. (B) Response of the Mx component to the Grad-3919-Grad sequence with an interpulse delay of 75 μs. The total length for the water suppression sequence was 2.4 ms (C) Response of the M_x component to a WATERGATE sequence (Grad-Sel90-Hard180-Sel90-Grad). The total length for the suppression sequence was 6.04 ms. (D) Response of the My component to the sequence Sel90-Hard90-Grad-Sel90-Hard180-Sel90-Grad. The total length of the suppression sequence was 8.03 ms. (E) Response of the Mx component to the excitation sculpting sequence (Grad1-Sel90-Hard180-Grad1-Grad2-Sel90-Hard180-Grad2). The total length of the water suppression sequence was 8.04 ms. (F) Response of the M_y component to the W1F90-Grad-WR4-Grad sequence. The total length of the water suppression sequence was 5.01 ms with a RF amplitude of the WADE pulses of 5 kHz. Selective pulses used in the WATERGATE sequences were Sinc pulses of 2 ms and a RF amplitude for the hard pulse of 25 kHz. The contour lines in the plot are shown at a fidelity level of 0.95.

Figure 3.

Transverse magnetization response as a function of the RF offset to various water suppression schemes used in 2D [¹H,¹H] NOESY experiments. For simplicity, the offset is reported in ppm. (A) 3–9-19 sequence with an interpulse delay of 200 μs. (B) 3–9-19 sequence with an interpulse delay of 75 μs. (C) WATERGATE sequence. (D) modified WATERGATE with water flip-back pulses. (E). Excitation sculpting sequence. (F) WADE water suppression sequence. The green lines show the M_z magnetization that on-resonance reaches a maximum for the WATERGATE with water flip-back (D) and WADE (F) sequences.

Figure 4.

Responses of the transverse magnetization to the different water suppression schemes used in the [¹H,¹H] NOESY experiments as a function of the RF amplitude. (A) and (B) 3–9-19 with an interpulse delay of 200 μs, and 75 μs, respectively. (C) WATERGATE sequence, (D) modified WATERGATE sequence (Lippens et al.). (E) excitation sculpting and (F) WADE-NOESY suppression. Both WATERGATE sequences (C and D) have non-zero transverse water magnetization with RF inhomogeneity.

Figure 5.

(A) [¹H,¹H] WADE-NOESY pulse sequence. The two non-filled rectangles are hard π/2 pulse, and the filled rectangles are WADE pulses. The phases φ₁, φ₂, φ₃, and the receiver phase (φ_2r) were {x, -x}, { x, x, x, x, x, x, x, x, -x, -x, -x, -x, -x, -x, -x, -x }, {x, x, -x, -x, -y, -y, y, y}, and { x, -x, -x, x, y, -y, -y, y, -x, x, x, -x, -y, y, y, -y} respectively. The state of water magnetization is given near each pulse. Starting with z magnetization on water (W_z), a mixture of transverse magnetization is created (aW_y + bW_x) during the t1 evolution. Here we assume no relaxation during the t1 delay. The value of a and b are a function of chemical shift and t₁. During the long mixing time the water magnetization returns to the z-direction. Both WADE pulses do not affect the state of water magnetization, which remains along the z-direction during acquisition. (B) Phase shape of the W1F90 pulse and corresponding excitation profile simulated with an initial magnetization M_z. (C) Phase shape of WR4 pulse and its simulated response with initial magnetizations M_x (red) and M_y (green). The amplitude for both pulses is kept constant at 6.33 kHz.

Figure 6.

(A) Comparison of the 2D [¹H-¹H] NOESY of UBI^K48C with a 150 ms mixing time using ES and WADE suppression scheme. The peaks in red are positive and those in black are negative. All experiments were performed on a Bruker 850 MHz spectrometer at 303 K using identical acquisition parameters: 256 number of complex points were acquired in indirect dimension and 8 scans per FID with a relaxation delay of 2 sec. Identical processing parameters were used for both spectra. (B) 1D slices of the 2D [¹H-¹H] ESNOESY (blue) and [¹H-¹H] WADE-NOESY (green) for the peaks highlighted in A. (C) NOESY buildup curves [¹H-¹H] ES-NOESY(blue) and [¹H-¹H] WADE-NOESY (green) for the resonances marked A.

Figure 7.

(A) [¹H-¹H] WADE-NOESY of UBI^K48C with WADE pulses of 3.33 kHz to improve peak detections near the water signal. The highlighted regions emphasize resonances with a significantly higher intensity than those in the ES-NOESY. The experiment was performed on a Bruker 850 MHz spectrometer at 303 K. 128 number of complex points were acquired in indirect dimension and 4 scans per FID with a relaxation delay of 2 sec. (B) 1D slices from near water and far off resonance regions of the spectrum.

Figure 8.

2D [¹H,¹H] NOESY spectra of RKIP with a 150 ms mixing time using (A) ES as a water suppression sequence and (B) WADE water suppression. Positive peaks are shown in red and negative peaks in black. All experiments were performed on a Bruker 900 MHz spectrometer at 300 K using identical acquisition parameters with 128 complex time points in the indirect dimension and 8 scans per FID. The relaxation delay was set to 2 sec. Both spectra were processed using NMRpipe with identical processing parameters. We have used the sine window function with offsets 0.2 and 0.45 in t2 and t1 dimensions, respectively. NMRpipe solvent filter (SOL) is used with a sine low pass filter. Zero filled the complex FID data to a final size of 1024*512 complex points. A baseline correction using 4th order polynomial is performed in both dimensions. (B) 1D slices were taken from the 2D ES-NOESY (blue) and WADE-NOESY (green) for the peaks highlighted in A.

Figure 9.

Comparison of cross peak intensities for the different pulse sequences. (A) Amide region of UBI^K48C. For simplicity, the cross-peaks were indicated with random numbers. Sensitivity gain of the NOE cross peaks of the 2D [¹H,¹H] WADE-NOESY relative to (B) presat-NOESY (noesyphpr). Red bars represent the peaks that broaden out due to exchange. (C) ES-NOESY (noesyesgpph), and (D) 3919-NOESY (noesygpph19). All experiments were recorded on Bruker 900 MHz spectrometer at 300K with 128 complex time points in indirect dimension and 8 scans per FID. A relaxation delay of 2 sec was used for all the experiments. The spectra were processed with identical processing parameters.