Enhanced Sensitivity of 1H,1H Nuclear Overhauser Effects using Water Irradiation Devoid Pulses

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 inter-actions. Since NMR studies of biomacromolecules are carried out in aqueous solutions, [1H,1H] NOESY pulse sequences must incorporate water suppression schemes to reduce the water signal that dominates 1H detected experiments. These pulse schemes are also designed to minimize signal 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 [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 (RKIP). The new pulse sequence shows a net increase of the NOE intensities varying from 30 to 170% compared to the commonly used NOESY experiments with excitation sculpting sequence as a water suppression element. The new WADE pulses can be easily implemented into 2D and 3D homo- and hetero-nuclear NOESY pulse sequences.