DIFFUSION FILTERS FOR SEPARATION OF SOLVENT-PROTEIN AND PROTEIN-PROTEIN NUCLEAR OVERHAUSER EFFECTS (HYDRA)

Nuclear magnetic resonance (NMR) spectroscopy in solution has a unique potential for providing novel insights into structural and dynamic as pects of the solvation of biological macromolecules such as proteins, which is based on the observation of intermolecular solvent-protein nu clear Overhauser effects (NOEs) in the laboratory frame and the rotati ng frame. In these experiments spectral overlap between resonance line s of the solvent and the macromolecule may make the distinction betwee n certain water-protein and protein-protein NOEs ambiguous even in two - and higher-dimensional NMR experiments. Here we show that use of dif fusion filters in NOE difference experiments enables the observation o f hydration water molecules without interference from intramolecular N OEs. Water-protein NOEs can thus be unambiguously identified from meas urements at a single set of solution conditions. The proposed approach works well also with very short mixing times, and the resulting spect ra are easy to analyze. One-dimensional experiments with diffusion fil ters have been combined with higher-dimensional experiments to obtain the chemical shift dispersion needed for individual resonance assignme nts.