Water-macromolecule interactions by NMR: A quadrature-free constant-time approach and its application to CI2

A pulse sequence is proposed to select water magnetization with enhanced specificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented without quadrature detection, which results in a √2 increase in signal-to-noise ratios as compared to traditional non-selective methods for water filtration. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gains in sensitivity. The proposed experiment was applied to unlabeled HEW lysozyme and to 15 N-labeled chymotrypsin inhibitor 2 which was partially or non 13C-enriched. Water molecules belonging to a spine of hydration between two pseudo β-sheet strands were identified, solving previously reported discrepancies between the X-ray and the refined NMR structure of CI2. The proposed experiment is particularly suitable for hydration studies of mixtures of labeled and unlabeled components, such as ligand–macromolecule complexes.