Optimization of reverse NOE pumping experiments in the analysis of complex systems with densely crowded NMR spectra

We present an optimization of Reverse NOE-pumping (RNP) in order to observe the ¹H signals of ligands bound to proteins. Although various ligand-based NMR screening methods have been proposed, the most frequently used method has been Saturation-Transfer Difference (STD), owing to the relatively easy setup of experiments. Yet the critical point of STD is the selective irradiation of protein without irradiating ligand, and thus the STD technique is unable to observe ¹H ligand signals, which resonate across the entire ¹H spectral width. In the present study, the RNP experiment has been improved to develop an effective NMR-based screening technique. The optimized RNP spectra reveal less subtraction artifacts and phase distortion than the original RNP spectra, indicating its applicability to any type of ligand molecules.