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

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.


Introduction
Since its discovery in the early 50s, the nuclear Overhauser effect (NOE) has been an essential parameter for NMR characterization of biomacromolecules (Overhauser 1953;Carver and Slichter 1953;Kumar et al. 1980). NOE results from the dipolar interactions between two nuclear spins, and its dependence on the internuclear distances has enabled the structure determination of biomacromolecules (Kumar et al. 1980(Kumar et al. , 1981Wuthrich 1986;Vögeli 2014). The introduction of the transverse relaxation optimized spectroscopy (TROSY) experiments (Pervushin et al. 1997) has furthered the importance of NOEs, expanding the application of NMR spectroscopy to larger proteins and protein complexes (Pervushin et al. 1999). More recently, methyl-methyl NOE has emerged as a critical tool for site-specific assignments of side-chain methyl groups in proteins, enabling the structural and dynamic characterization of sizeable supramolecular assemblies (Zwahlen et al. 1998). Finally, a thorough quantitation of NOE (exact NOE or eNOE) (Vögeli 2014;Vögeli et al. 2016) has also led to the identification of multiple conformational states of proteins (Vögeli et al. 2016) as well as intra-molecular allosteric pathways (Strotz et al. 2020).
All biologically relevant NMR studies are performed in aqueous solutions with a concentration of the biomacromolecules less than 1 mM and a proton concentration from the water of 110 M. The latter has represented a long-standing challenge for the receivers of the NMR spectrometers, especially for 1 H-detected experiments (Price 1999;Zheng and Price 2010). Typical homo-and heteronuclear NOE-based pulse sequences, however, are equipped with water suppression schemes, which minimize the water signal and detect [ 1 H, 1 H] NOE correlations at high sensitivity. Some of these pulse schemes also reduce the unwanted exchange between labile sites (e.g., amide protons) and water, increasing the intensity of NOE cross peaks. In addition, high and ultrahigh magnetic fields enhance the longitudinal relaxation of the spin magnetization and augment NOE sensitivity; yet, radiation damping complicates water suppression (Krishnan and Murali 2013;Warren and Richter 2007).
The most common water-suppression schemes used for NOE measurements include selective solvent presaturation, spin-lock pulses, jump-return sequences, selective defocusing of the water signal, and shaped pulses (Nguyen et al. 2007;Liu et al. 1998;Hwang and Shaka 1995;Chen et al. 2017;Hoult 1976). Other methods are based on the differential relaxation time or diffusion properties between the water and biomolecules (Price 1999;Zheng and Price 2010). One of the most effective schemes is water presaturation (presat), which consists of a weak continuous radio frequency (RF) field that irradiates the water signal for 1-2 s before executing the pulse sequence (Hoult 1976). Nonetheless, the weak RF field attenuates the proton resonances in rapid exchange with solvent, e.g., amide protons that constitute proteins' fingerprint, and the effectiveness of solvent presaturation is significantly reduced by spin diffusion (Grzesiek and Bax 1993). The WATERGATE sequence avoids these drawbacks (Piotto et al. 1992). WATERGATE is a pulsed gradient spin echo (PGSE) (Stejskal and Tanner 1965) sequence with the hard π pulse replaced by the sequence 'selective π/2-Hard π-selective π/2', which inverts all signals except the solvent. For solute resonances, the effect of the first gradient pulse is reversed by the second gradient with a π pulse applied to the solute resonances. As a result, the dephasing effect of the two gradient pulses is accumulated, and the solvent signal is suppressed. However, the sequence of selective and hard pulses in WATERGATE can cause spectral phase distortions, a problem that was solved using improved versions of WATERGATE (Liu et al. 1998;Adams et al. 2013) or the excitation sculpting (ES) sequence (Stott et al. 1995). Among these sequences, ES is inhomogeneity compensated and its water selectivity and irradiation bandwidth can be easily tailored. Binomial (1-1, 1-3-3-1, etc.) (Hore 1983a(Hore , 1983b or binomial-like (3-9-19 or W5) (Sklenar et al. 1993) sequences have also been used for water suppression in 2D and 3D NOESY experiments (Marion et al. 1989). These symmetric schemes feature a π-shifted phase in the second half of the sequence. When the RF carrier is set on resonance with the water signal, the effective operation of the right half is canceled by the left half of the sequence, and the solute coherences are refocused and detected. Similar to WATERGATE, the solvent resonances in the binomial sequences are suppressed by a pair of dephasing gradients.
Notably, the reduced saturation transfer from water to the solute makes binomial sequences favored over water presaturation. Nevertheless, the binomial sequences have limited bandwidth irradiation,which represents a problem for biomolecules with broad chemical shift dispersion. Ideally, for biomolecular NMR spectroscopy at high and ultra-high magnetic fields, a water-suppression scheme should have high selectivity for the water signal, broadband irradiation, high fidelity of spin operations, and should be compensated for the different sources of inhomogeneity.
Recently, we developed a new software, GENErator of TrIply Compensated pulSes via Artificial Intelligence or GENETICS-AI, that combines an evolutionary algorithm and artificial intelligence (AI) to create RF pulses with constant amplitude and variable phase shapes (Manu et al. 2022b). Using GENETICS-AI, we generated broadband pulses (called WADE for Water irrAdiation DEvoid pulses) that elude water irradiation while exciting a large bandwidth at high-fidelity (Manu et al. 2022a). The WADE pulses used for water-suppression increase the sensitivity of the transverse relaxation optimized (TROSY) [ 1 H, 15 N] HSQC pulse sequence (Manu et al. 2022a). Here, we report the implementation of WADE-π/2 (W1F90) and WADE-π (WR4) pulses into a standard 2D [ 1 H, 1 H] NOESY experiment (i.e., [ 1 H, 1 H] WADE-NOESY) and show that they significantly enhance the NOE cross peaks intensities for labile, and to a lesser extent, non-labile proton resonances of two wellfolded proteins: the K48C mutant of ubiquitin (UBI K48C ) and Raf kinase inhibitor protein (RKIP). We show that the intensities of the NOESY cross-peaks follow the typical kinetics of the NOE buildup, with an increase of the crosspeak intensities ranging from 30 to 170%. The WADE pulses can be easily implemented into homo-and hetero-nuclear NOESY-type of experiments to boost their sensitivity.

Expression and purification of ubiquitin K48C mutant
Uniformly 15 N labeled K48C ubiquitin mutant (UBI K48C ) was expressed and purified as reported by Olivieri et al. (Olivieri et al. 2018). Briefly, the recombinant human K48C mutant was generated from the ubiquitin wild-type gene (pRSET) vector using the QuickChange® kit from Stratagene (CA, USA). The protein was expressed in E. coli BL21 (DE3) cells and cultured at 30 °C in M9 minimal media, containing 15 NH 4 Cl (Cambridge Isotope Laboratories Inc.) salt as the only nitrogen source. Protein overexpression was induced at an optical density (OD 600 ) above 0.8 by adding 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) and was carried out for 5 h. The cell suspension was centrifuged at 6370 g for 30 min at 4 °C, and the cell pellet was collected, flash-frozen, and stored at -20 °C. For protein purification, the cell pellet was resuspended in 50 mL of 50 mM sodium acetate (pH 5.0) and 5 mM of β-mercaptoethanol (β-me) buffer, homogenized with a cell grinder, and sonicated using Branson Sonifier 450 (output of 4; duty cycle, 40%) for 10 min. Cell debris was pelleted by centrifugation at 45,700 g for 40 min at 4 °C. The pooled supernatant UBI K48C was loaded into a column containing a Whatman P11 phosphocellulose resin (Sigma-Aldrich) and UBI K48C was eluted with a NaCl gradient from 0-1 M. The fractions containing UBI K48C were concentrated and loaded into a column of Sephacryl S-100 resin (GE Healthcare) using 100 mM phosphate buffer at pH 7.0 as a mobile phase. The purified protein was concentrated, lyophilized, and stored at -20 °C. For NMR samples, 15 N UBI K48C was solubilized in 10 mM sodium acetate buffer (pH 6.0), 100 mM NaN 3 , and 2 mM dithiothreitol (DTT) to final protein concentration of 0.5 mM.

Expression and purification of RKIP
Recombinant Raf kinase inhibitor protein (RKIP) was expressed and purified as reported previously by Lee et al. (Lee et al. 2022). Transformed E. coli BL21(DE3) pLysS cells (Invitrogen™) were grown in M9 minimal media containing 15 NH 4 Cl (Cambridge Isotope Laboratories Inc.) at 30 °C. Protein overexpression was induced by adding 0.4 mM IPTG when the OD 600 was above 1.1 and was carried out for 5 h. The cells were harvested by centrifugation at 6370 g for 30 min at 4 °C, and the cell pellet was collected and stored at -20 °C. The cell pellet was then resuspended in 35 mL of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20% sucrose, 0.15 mg/ml lysozyme, 1 tablet of protease inhibitor (cOmplete™, Roche Applied Science), 100 U/mL DNAse I (Roche Applied Science), and 5 mM β-me and lysed using French press at 1000 psi. Cell debris was by centrifugation at 45,700 g for 40 min at 4 °C, and the supernatant was batchbound with Ni 2+ -NTA agarose affinity resin (Thermofisher) at 4 °C for 3 h or overnight. The resin/proteins mixture was loaded into a column and washed using 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20% sucrose, 1 mM PMSF, 5 mM β-me. The protein was eluted in two fractions using the same buffer supplied with 200 mM and 500 mM of imidazole, respectively. Since the RKIP gene was cloned into a pET SUMO vector (Invitrogen™), the His-tagged-SUMO was removed using a stoichiometric amount of recombinant UPL1 protease in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM β-me, 0.5 mM PMSF. The cleavage reaction was performed overnight at 4 °C. A Ni 2+ -NTA purification was performed to eliminate contaminants. The fractions containing the un-tagged RKIP (flow-through) were concentrated using a 10 kDa concentrator (MilliporeSigma, Life Science) and loaded into a 16/60 Hi Load Superdex 200 (GE Healthcare) size exclusion column (SEC) using 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM DTT as a mobile phase. The purified protein was concentrated and stored at 4 °C in the SEC buffer supplied with protease inhibitors. 15 N labeled RKIP sample was prepared by buffer-exchanging and concentrating the stored protein in 20 mM KH 2 PO 4 (pH 6.5), 10 mM DTT, 10 mM MgCl 2 , and 1 mM NaN 3 buffer supplied with 5% D 2 O, and 0.5% Pefa block® (Sigma-Aldrich, USA) to a final protein concentration of 0.3 mM.

NMR spectroscopy
NMR experiments were performed on an 850 MHz Bruker Avance NEO equipped with a TCI cryoprobe. Ten mixing times were used for the NOESY buildup curves: 25,50,75,100,150,200,300,400,500, and 700 ms. The new WADE pulses were implemented into the NOESY experiments available from the Bruker library (noesyfpgpph19/n oesyesgpph), where the third π/2 pulse was replaced by a WADE-π/2 (W1F90) pulse, and the WATERGATE 3919 was replaced by the WADE-π (WR4) pulse. Note that the names of the WADE pulses is reported according to the nomenclature used in our recent paper on GENETICS-AI (Manu et al. 2022b). The acquisition parameters consisted of 1024 points in the direct dimension and 256 complex points in the indirect dimension. All spectra were processed using NMRPipe (Delaglio et al. 1995). Both dimensions were processed using a sine bell window function shifted by 90° and zero-filled to double the size for a final matrix of 1024 × 512.

Results and discussion
We first evaluated the performance of the most common water suppression schemes implemented in the 2D [ 1 H, 1 H] NOESY pulse sequences of the Bruker library: Grad-3919-Grad, WATERGATE (Grad-Sel90-Hard180-Sel90-Grad), WATERGATE with flip-back pulses (Sel90-Hard90-Grad-Sel90-Hard180-Sel90-Grad) (Lippens et al. 1995), and ES pulses (Grad1-Sel180-Hard180-Grad1-Grad2-Sel180-Hard180-Grad2) (Stott et al. 1995). Figure 1 shows the schematics of these pulse sequences and the resulting operators for both water and protein magnetization. We simulated the responses of the transverse magnetization (M x or M y ) to the different water-suppression schemes for a bandwidth of 20 kHz varying the RF offset and amplitude (B 1 ± 20%, Fig. 2). As expected, the Grad-3919-Grad sequence shows a periodic response of M x within the bandwidth considered.
Using an interpulse delay of 200 μs, we obtained an excitation bandwidth relatively narrow (see the areas with fidelity levels of 0.5, Fig. 2A). Note that the bandwidth covered by the Grad-3919-Grad sequence can be tailored by changing the interpulse delay, as shown in Fig. 2B, though a trade-off is necessary between the bandwidth irradiated and water selectivity. We then analyzed the responses of the transverse components of the magnetization for the WATERGATE and the WATERGATE with water flip-back pulse sequences (Fig. 2C, D). Unlike the 3919 sequences, the WATERGATE sequences display an improved water selectivity and broader irradiation regions with a fidelity level of 0.95. Finally, we tested the ES sequence, which showed a better water selectivity, although its high-fidelity region is somewhat reduced (Fig. 2E). Overall, the fidelity of the magnetization response for all the water suppression schemes considered has a limited bandwidth, spanning approximately 10 ppm with no scaling of the B 1 field.
With this in mind, we programmed GENETICS-AI (Manu et al. 2022b) to create high-fidelity constant-amplitude pulses that elude water irradiation and achieve broader bandwidths than the classical water-suppression schemes. GENETICS-AI utilizes an evolutionary algorithm to generate a library of optimal phase shapes (OPS) to train an artificial intelligence (AI) module of MatLab® and create optimal solutions to a specific problem (Manu et al. 2022b).
The WADE pulses designed by GENETICS-AI excite a significantly broader bandwidth with a higher level of fidelity relative to the previous pulse schemes and do not irradiate the water signal (Manu et al. 2022a). The response of the transverse magnetization to the WADE pulse scheme is reported in Fig. 2F. For clarity, we also present the 1D profiles of the simulated magnetization responses by changing RF offset (Fig. 3) and amplitude (Fig. 4). Note that the sign of the M y response for WR4 pulse is inverted to obtain null irradiation on resonance (water signal), leaving the z-component of the water signal virtually unperturbed (Fig. 3F). As a result, the spectrum will display resonances with a positive sign for values of chemical shifts greater than 4.7 ppm and a negative sign for values less than 4.7 ppm. A detailed comparison of the WADE pulse' performance with other binomial like sequences is reported in our previous article (Manu et al. 2022a).
We then implemented the W1F90 and WR4 pulses into the 2D [ 1 H, 1 H] NOESY pulse sequence (Fig. 5). The general scheme was taken from the noesygpph19 code available in the Bruker sequence library. We did not change the first two hard π/2 pulses that excite the entire 1 H bandwidth, D The excitation sculpting sequence with a total duration of 8.04 ms. 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 1 H channel represent water-selective π/2 or π pulses. Filled shapes represent gradient pulses of 1 ms duration including the water signal. We substituted, however, the hard π/2 pulse after the mixing time with a W1F90 pulse (red) and the water-suppression scheme with a WR4 pulse (blue), which is flanked with two gradient pulses along the z-direction to remove spurious magnetization. The W1F90 pulse excites the left and right sides of the bandwidth with high fidelity, leaving a null excitation on-resonance. The M y component of the magnetization as a function of the RF offset follows a sigmoidal behavior, with an on-resonance point of inversion. The latter is accomplished by modulating the phase shape, as indicated in Fig. 5B. The WR4 pulse for the coherence detection is the same utilized for our previously published [ 1 H, 15 N] TROSY-HSQC sequence (Manu et al. 2022a). The WR4 pulse does not affect the M x component of the magnetization and operates an inversion operation on both sides of the bandwidth, avoiding water irradiation.
To experimentally assess the performance of the WADE pulses, we compared the 2D [ 1 H, 1 H] NOESY experiment with ES water suppression (Bruker sequence noesyesgpph) with the [ 1 H, 1 H] WADE-NOESY pulse sequence UBI K48C . A comparison of the 2D NOESY spectra is shown in Fig. 6A. The UBI K48C fold comprises a five-stranded β sheet, an α helix, and a short 3 10 helix; therefore, a significant number of NOEs are expected. The peaks of the [ 1 H, 1 H] WADE-NOESY spectrum have a negative sign in the range of chemical shifts from -1.0 to 4.7 ppm and a positive sign from 4.7 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 WATER-GATE sequences were Sinc pulses of 2 ms and an RF amplitude for the hard pulse of 25 kHz. The contour lines in the plot are shown at a fidelity level of 0.95 to 10 ppm, according to the W1F90 pulse excitation profile of Fig. 5 (Fig. 6B). We also observed an enhancement of the resonance intensities for the NOE cross-peaks in the aliphatic region of the protein.
In this case, the signal enhancement can be attributed to the higher fidelity of the spin operations for the two WADE pulses, especially for peaks at the two edges of the spectrum. Overall, we observe an increase in signal intensity Fig. 3 Transverse magnetization response as a function of the RF offset to various water suppression schemes used in 2D [ 1 H, 1 H] 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 Modi-fied 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 of ~ 50% for these resonances. We then tested the NOE buildup for several resonances as a function of the mixing time (Fig. 6C). From the selected residues, it is possible to appreciate the typical kinetics of the NOE buildup and the significant enhancement of the NOE cross peaks intensities relative to the [ 1 H, 1 H] ES-NOESY spectra. While the peak intensities are enhanced throughout the spectrum, the peak intensities near the water signal are weaker than the corresponding cross peaks in the [ 1 H, 1 H] ES-NOESY spectrum. The latter can be easily overcome by increasing the water selectivity, i.e., reducing the WADE pulses amplitude. This is demonstrated in Fig. 7, which shows the [ 1 H, 1 H] WADE-NOESY spectrum obtained by lowering the WADE pulse amplitude to 3.33 kHz. In this case, the peaks near the water signals are significantly more intense than the experiment performed with the ES sequence. Also, the broader irradiation bandwidth of the WADE pulses enhances the peak intensities at both edges of the spectrum relative to the ES sequence (see highlighted regions of Fig. 7).
We also compared the performance of the two pulse sequences for RKIP, a well-folded protein involved in kinase signaling (Lee et al. 2022). As for UBI K48C , the number of NOE cross-peaks obtained for RKIP with the [ 1 H, 1 H] WADE-NOESY experiment is substantially higher than the Fig. 4 Responses of the transverse magnetization to the different water suppression schemes used in the [ 1 H, 1 H] 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. 1995). E excitation sculpting and F WADE-NOESY suppression. Both WATER-GATE sequences (C and D) have a non-zero transverse water magnetization with B 1 scaling corresponding [ 1 H, 1 H] ES-NOESY spectrum (Fig. 8A). The latter is even more apparent for peaks at the two edges of the bandwidth, as illustrated in the cross-sections of Fig. 8B, where some of the cross-peaks are barely detectable with the [ 1 H, 1 H] ES-NOESY experiment. In addition, a significant increase in the number and intensity of the cross-peaks is noticeable for the H N -H N region.
To determine more quantitatively the signal enhancement of the [ 1 H, 1 H] WADE-NOESY over the other pulse schemes of Fig. 1, we calculated the relative gain in sensitivity. Figure 9A shows the amide region of UBI K48C with the H N -H N cross-peaks indicated by random numbers. The histograms in Fig. 9B-D show the differences in the intensities of the cross-peaks relative to the [ 1 H, 1 H] WADE-NOESY The two non-filled rectangles are the hard π/2 pulse, and the filled rectangles are the WADE pulses. The phases φ 1 , φ 2 , φ 3 , and the receiver phase (φ r ) 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 t 1 evolution. Here we assume no relaxation during the t 1 delay. The value of a and b are a function of the chemical shift and t 1 . During the long mixing time the water magnetization returns to the z-direction. Both the 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 of both pulses is kept constant at 6.33 kHz experiment. Compared to the [ 1 H, 1 H] NOESY with presaturation (presat-NOESY), the [ 1 H, 1 H] WADE-NOESY spectrum shows an average increase of cross-peak intensity of ~ 60%. The highest gain (> 200%) is achieved for labile H N resonances that broaden out partially or entirely due to the exchange with the water signal caused by the weak RF field used in the presaturation sequence. The comparison with ES and 3919 sequences shows an average sensitivity gain of 89% and 30%, respectively. In these latter cases, the higher fidelity of operations associated with the WADE pulses may explain the signal enhancement.
RF pulse design is fundamental for developing NMR spectroscopy at high and ultra-high magnetic fields (Ardenkjaer-Larsen et al. 2015). In the past years, many research groups have contributed to designing and developing new pulses or pulse sequences that enhance NMR signal detection for biological macromolecules (Schanda 2009;Guéron et al. 1991;Rance et al. 1999;Zhu et al. 1999). However, NMR spectroscopy at ultra-high magnetic fields calls for RF pulses that irradiate larger bandwidths, with high fidelity of spin operations and compensation for instrumental inhomogeneity. We recently showed that a combination of an evolutionary algorithm with AI enables the design of new RF pulses with a constant amplitude to excite broader bandwidth (Manu et al. 2022b). These new pulses can be used for a variety of applications, spanning from spin entanglement to solution (Manu et al. 2022b(Manu et al. , 2022a and solid-state (Gopinath et al. 2022) NMR spectroscopy, as well as magnetic resonance imaging (Manu et al. 2022b). The design of the WADE pulses is only an example of the new possibilities that the new GENETICS-AI software opened up.
In conclusion, we presented the implementation of W1F90 and WR4 pulses in the 2D [ 1 H, 1 H] NOESY pulse Fig. 7 A [ 1 H-1 H] WADE-NOESY of UBI K48C with WADE pulses of amplitude 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 complex points in indirect dimension and 4 scans per FID were acquired with a relaxation delay of 2 s. B 1D slices from near water and far off resonance regions of the spectrum sequences, demonstrating a significant enhancement in the detection of internuclear NOE. The enhancement is due to the broadband nature of the new pulses and a substantially reduced magnetization exchange between water and labile amide protons. Concomitantly, we observed an increase in sensitivity of NOEs for non-exchangeable protons, which is due to the higher fidelity spin operation of the WADE pulses relative to the commonly used water suppression schemes. We anticipate that the WADE pulses will improve the sensitivity of the recently developed L-PROSY experiments (Novakovic et al. 2018) for labile protons as well as heteronuclear 3D 15 N-or 13 C-edited NOESY experiments that are more suitable for larger biomacromolecules. 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 s. Both spectra were processed using NMRpipe with identical processing parameters. Sine window functions with offsets of 0.2 and 0.45 were used in the t 2 and t 1 dimensions, respectively. NMRpipe solvent filter (SOL) was used with a sine low pass filter. The complex FID data were zero-filled to a final size of 1024*512 complex points. A baseline correction using 4th order polynomial was performed in both dimensions. B 1D slices were taken from the 2D ES-NOESY (blue) and WADE-NOESY (green) spectra for the peaks highlighted in A  Fig. 9 Comparison of NOE cross peak intensities of the amide region of RKIP for the different pulse sequences. A Spectrum of the amide region of UBI K48C . For simplicity, the cross-peaks were indicated with random numbers. B Sensitivity gain of the NOE cross peaks obtained with the 2D [ 1 H, 1 H] WADE-NOESY experiment relative to presat-NOESY (noesyphpr). Red bars represent the peaks that broaden out due to exchange. C Sensitivity gain relative to the ES-NOESY (noesyesgpph) experiment. D Sensitivity gain relative to the 3919-NOESY (noesygpph19) experiment. All experiments were recorded on a Bruker 900 MHz spectrometer at 300 K with 128 complex time points in the indirect dimension and 8 scans per FID. A relaxation delay of 2 s was used for all the experiments. The spectra were processed with identical processing parameters