Investigation of dipolar-mediated water-protein interactions in microcrystalline Crh by solid-state NMR spectroscopy.

Water-protein interactions play a major role in protein folding, structure, and function, and solid-state NMR has recently been shown to be a powerful tool for the site-resolved observation of these interactions in solid proteins. In this article we report investigations on possible water-protein dipolar transfer mechanisms in the microcrystalline deuterated protein Crh by a set of solid-state NMR techniques. Double-quantum (DQ) filtered and edited heteronuclear correlation experiments are used to follow direct dipolar water-protein magnetization transfers. Experimental data reveal no evidence for "solid-like" water molecules, indicating that residence times of solvent molecules are shorter than required for DQ creation, typically a few hundred microseconds. An alternative magnetization pathway, intermolecular cross-relaxation via heteronuclear nuclear Overhauser effects (NOEs), is probed by saturation transfer experiments. The significant additional enhancements observed when irradiating at the water frequency can possibly be attributed to direct heteronuclear water-protein NOEs; however, a contribution from relayed magnetization transfer via chemical exchange or proton-proton dipolar mechanisms cannot be excluded.