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 sp ecificity through a synergetic combination of several filtering principles. This approach relies on a constant-time evolution period implemented witho ut quadrature detection, which results in a root 2 increase in signal-to-no ise ratios as compared to traditional non-selective methods for water filtr ation. In addition, the quadrature-free constant-time block facilitates the implementation of the water flip-back strategy, which leads to further gai ns in sensitivity. The proposed experiment was applied to unlabeled HEW lys ozyme and to N-15-labeled chymotrypsin inhibitor 2 which was partially or n on C-13-enriched. Water molecules belonging to a spine of hydration between two pseudo beta-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 mixtu res of labeled and unlabeled components, such as ligand-macromolecule compl exes.