Water irradiation devoid pulses enhance the sensitivity of 1 H, 1 H 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 [ 1 H, 1 H] NOESY pulse sequences incorporate water suppression schemes to reduce the water signal that dominates 1 H-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 [ W ater irr A diation DE void (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 [ 1 H, 1 H] 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 [ 1 H, 1 H] 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.